Compositions for modulating expression of c9orf72 antisense transcript

ABSTRACT

Disclosed herein are compositions and methods for reducing expression of C9ORF72 antisense transcript in an animal with C9ORF72 antisense transcript specific inhibitors. Such methods are useful to treat, prevent, or ameliorate neurodegenerative diseases in an individual in need thereof. Such C9ORF72 antisense transcript specific inhibitors include antisense compounds.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0262USD1SEQ_ST25.txt created Aug. 24, 2020, which is 76 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD

Provided are compositions and methods for inhibiting expression ofC9ORF72 antisense transcript in an animal. Such compositions and methodsare useful to treat, prevent, or ameliorate neurodegenerative diseases,including amyotrophic lateral sclerosis (ALS), frontotemporal dementia(FTD), corticobasal degeneration syndrome (CBD), atypical Parkinsoniansyndrome, and olivopontocerebellar degeneration (OPCD).

BACKGROUND

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative diseasecharacterized clinically by progressive paralysis leading to death fromrespiratory failure, typically within two to three years of symptomonset (Rowland and Shneider, N. Engl. J. Med., 2001, 344, 1688-1700).ALS is the third most common neurodegenerative disease in the Westernworld (Hirtz et al., Neurology, 2007, 68, 326-337), and there arecurrently no effective therapies. Approximately 10% of cases arefamilial in nature, whereas the bulk of patients diagnosed with thedisease are classified as sporadic as they appear to occur randomlythroughout the population (Chio et al., Neurology, 2008, 70, 533-537).There is growing recognition, based on clinical, genetic, andepidemiological data, that ALS and frontotemporal dementia (FTD)represent an overlapping continuum of disease, characterizedpathologically by the presence of TDP-43 positive inclusions throughoutthe central nervous system (Lillo and Hodges, J. Clin. Neurosci., 2009,16, 1131-1135; Neumann et al., Science, 2006, 314, 130-133).

To date, a number of genes have been discovered as causative forclassical familial ALS, for example, SOD1, TARDBP, FUS, OPTN, and VCP(Johnson et al., Neuron, 2010, 68, 857-864; Kwiatkowski et al., Science,2009, 323, 1205-1208; Maruyama et al., Nature, 2010, 465, 223-226; Rosenet al., Nature, 1993, 362, 59-62; Sreedharan et al., Science, 2008, 319,1668-1672; Vance et al., Brain, 2009, 129, 868-876). Recently, linkageanalysis of kindreds involving multiple cases of ALS, FTD, and ALS-FTDhad suggested that there was an important locus for the disease on theshort arm of chromosome 9 (Boxer et al., J. Neurol. Neurosurg.Psychiatry, 2011, 82, 196-203; Morita et al., Neurology, 2006, 66,839-844; Pearson et al. J. Nerol., 2011, 258, 647-655; Vance et al.,Brain, 2006, 129, 868-876). This mutation has been found to be the mostcommon genetic cause of ALS and FTD. It is postulated that the ALS-FTDcausing mutation is a large hexanucleotide (GGGGCC) repeat expansion inthe first intron of the C9ORF72 gene (Renton et al., Neuron, 2011, 72,257-268; DeJesus-Hernandez et al., Neuron, 2011, 72, 245-256). A founderhaplotype, covering the C9ORF72 gene, is present in the majority ofcases linked to this region (Renton et al., Neuron, 2011, 72, 257-268).This locus on chromosome 9p21 accounts for nearly half of familial ALSand nearly one-quarter of all ALS cases in a cohort of 405 Finnishpatients (Laaksovirta et al, Lancet Neurol., 2010, 9, 978-985).

There are currently no effective therapies to treat suchneurodegenerative diseases. Therefore, it is an object to providecompositions and methods for the treatment of such neurodegenerativediseases.

Summary

Provided herein are compositions and methods for modulating levels ofC9ORF72 antisense transcript in cells, tissues, and animals. In certainembodiments, C9ORF72 antisense transcript specific inhibitors modulateexpression of C9ORF72 antisense transcript. In certain embodiments,C9ORF72 antisense transcript specific inhibitors are nucleic acids,proteins, or small molecules.

In certain embodiments, modulation can occur in a cell or tissue. Incertain embodiments, the cell or tissue is in an animal. In certainembodiments, the animal is a human. In certain embodiments, C9ORF72antisense transcript levels are reduced. In certain embodiments, C9ORF72antisense transcript associated RAN translation products are reduced. Incertain embodiments, the C9ORF72 antisense transcript associated RANtranslation products are poly-(proline-alanine),poly-(proline-arginine), and poly-(proline-glycine). In certainembodiments, the C9ORF72 antisense transcript contains a hexanucleotiderepeat expansion. In certain embodiments, the hexanucleotide repeat istranscribed in the antisense direction from the C9ORF72 gene. In certainembodiments, the hexanucleotide repeat expansion is associated with aC9ORF72 associated disease. In certain embodiments, the hexanucleotiderepeat expansion is associated with a C9ORF72 hexanucleotide repeatexpansion associated disease. In certain embodiments, the hexanucleotiderepeat expansion comprises at least 30 GGCCCC, CCCCCC, GCCCCC, and/orCGCCCC repeats. In certain embodiments, the hexanucleotide repeatexpansion comprises more than 30 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCCrepeats. In certain embodiments, the hexanucleotide repeat expansioncomprises more than 100 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC repeats.In certain embodiments, the hexanucleotide repeat expansion comprisesmore than 500 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC repeats. In certainembodiments, the hexanucleotide repeat expansion comprises more than1000 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC repeats. In certainembodiments, the hexanucleotide repeat expansion is associated withnuclear foci. In certain embodiments, C9ORF72 antisense transcriptassociated RAN translation products are associated with nuclear foci. Incertain embodiments, the antisense transcript associated RAN translationproducts are poly-(proline-alanine) and/or poly-(proline-arginine). Incertain embodiments, the compositions and methods described herein areuseful for reducing C9ORF72 antisense transcript levels, C9ORF72antisense transcript associated RAN translation products, and nuclearfoci. Such reduction can occur in a time-dependent manner or in adose-dependent manner.

Also provided are methods useful for preventing, treating, ameliorating,and slowing progression of diseases and conditions associated withC9ORF72. In certain embodiments, such diseases and conditions associatedwith C9ORF72 are neurodegenerative diseases. In certain embodiments, theneurodegenerative disease is amyotrophic lateral sclerosis (ALS),frontotemporal dementia (FTD), corticobasal degeneration syndrome (CBD),atypical Parkinsonian syndrome, and olivopontocerebellar degeneration(OPCD).

Such diseases and conditions can have one or more risk factors, causes,or outcomes in common. Certain risk factors and causes for developmentof a neurodegenerative disease, and, in particular, ALS and FTD, includegenetic predisposition and older age.

In certain embodiments, methods of treatment include administering aC9ORF72 antisense transcript specific inhibitor to an individual in needthereof. In certain embodiments, the C9ORF72 antisense transcriptspecific inhibitor is a nucleic acid. In certain embodiments, thenucleic acid is an antisense compound. In certain embodiments, theantisense compound is an antisense oligonucleotide. In certainembodiments, the antisense oligonucleotide is complementary to a C9ORF72antisense transcript. In certain embodiments, the antisenseoligonucleotide is a modified antisense oligonucleotide.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Additionally, as used herein, the use of “and” means “and/or” unlessstated otherwise. Furthermore, the use of the term “including” as wellas other forms, such as “includes” and “included”, is not limiting.Also, terms such as “element” or “component” encompass both elements andcomponents comprising one unit and elements and components that comprisemore than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this disclosure,including, but not limited to, patents, patent applications, publishedpatent applications, articles, books, treatises, and GENBANK AccessionNumbers and associated sequence information obtainable through databasessuch as National Center for Biotechnology Information (NCBI) and otherdata referred to throughout in the disclosure herein are herebyexpressly incorporated by reference for the portions of the documentdiscussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-OCH₂CH₂—OCH₃ and MOE) refers toan 0-methoxy-ethyl modification of the 2′ position of a furanose ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means anucleoside comprising a MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituentat the 2′-position of the furanose ring other than H or OH. In certainembodiments, 2′-substituted nucleosides include nucleosides withbicyclic sugar modifications.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“About” means within ±7% of a value. For example, if it is stated, “thecompounds affected at least about 70% inhibition of C9ORF72 antisensetranscript”, it is implied that the C9ORF72 antisense transcript levelsare inhibited within a range of 63% and 77%.

“Administered concomitantly” refers to the co-administration of twopharmaceutical agents in any manner in which the pharmacological effectsof both are manifest in the patient at the same time. Concomitantadministration does not require that both pharmaceutical agents beadministered in a single pharmaceutical composition, in the same dosageform, or by the same route of administration. The effects of bothpharmaceutical agents need not manifest themselves at the same time. Theeffects need only be overlapping for a period of time and need not becoextensive.

“Administering” means providing a pharmaceutical agent to an animal, andincludes, but is not limited to administering by a medical professionaland self-administering.

“Amelioration” refers to a lessening, slowing, stopping, or reversing ofat least one indicator of the severity of a condition or disease. Theseverity of indicators may be determined by subjective or objectivemeasures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antibody” refers to a molecule characterized by reacting specificallywith an antigen in some way, where the antibody and the antigen are eachdefined in terms of the other. Antibody may refer to a complete antibodymolecule or any fragment or region thereof, such as the heavy chain, thelight chain, Fab region, and Fc region.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein productencoded by such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding. Examples of antisense compounds include single-stranded anddouble-stranded compounds, such as, antisense oligonucleotides, siRNAs,shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels inthe presence of an antisense compound complementary to a target nucleicacid compared to target nucleic acid levels or in the absence of theantisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridizationof a compound with a target nucleic acid, wherein the outcome or effectof the hybridization is either target degradation or target occupancywith concomitant stalling of the cellular machinery involving, forexample, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise basepairing of nucleobases of an antisense oligonucleotide withcorresponding nucleobases in a target nucleic acid (i.e.,hybridization), and is mediated by Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar” means a furanose ring modified by the bridging of twoatoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleoside” (also BNA) means a nucleoside having a sugarmoiety comprising a bridge connecting two carbon atoms of the sugarring, thereby forming a bicyclic ring system. In certain embodiments,the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring.

“C9ORF72 antisense transcript” means transcripts produced from thenon-coding strand (also antisense strand and template strand) of theC9ORF72 gene. The C9ORF72 antisense transcript differs from thecanonically transcribed “C9ORF72 sense transcript”, which is producedfrom the coding strand (also sense strand) of the C9ORF72 gene.

“C9ORF72 antisense transcript associated RAN translation products” meansaberrant peptide or di-peptide polymers translated through RANtranslation (i.e., repeat-associated, and non-ATG-dependenttranslation). In certain embodiments, the C9ORF72 antisense transcriptassociated RAN translation products are any of poly-(proline-alanine),poly-(proline-arginine), and poly-(proline-glycine).

“C9ORF72 antisense transcript specific inhibitor” refers to any agentcapable of specifically inhibiting the expression of C9ORF72 antisensetranscript and/or its expression products at the molecular level. Asused herein, “specific” means reducing or inhibiting expression ofC9ORF72 antisense transcript without reducing non-target transcript toan appreciable degree (e.g., a C9ORF72 antisense transcript specificinhibitor reduces expression of C9ORF72 antisense transcript, but doesnot reduce expression of C9ORF72 sense transcript to an appreciabledegree). C9ORF72 specific antisense transcript inhibitors includenucleic acids (including antisense compounds), siRNAs, aptamers,antibodies, peptides, small molecules, and other agents capable ofinhibiting the expression of C9ORF72 antisense transcript and/or itsexpression products, such as C9ORF72 antisense transcript associated RANtranslation products.

“C9ORF72 associated disease” means any disease associated with anyC9ORF72 nucleic acid or expression product thereof, regardless of whichDNA strand the C9ORF72 nucleic acid or expression product thereof isderived from. Such diseases may include a neurodegenerative disease.Such neurodegenerative diseases may include ALS and FTD.

“C9ORF72 foci” means nuclear foci comprising a C9ORF72 transcript. Incertain embodiments, a C9ORF72 foci comprises at least one C9ORF72 sensetranscript (herein “C9ORF72 sense foci”). In certain embodiments,C9ORF72 sense foci comprise C9ORF72 sense transcripts comprising any ofthe following hexanucleotide repeats: GGGGCC, GGGGGG, GGGGGC, and/orGGGGCG. In certain embodiments, a C9ORF72 foci comprises at least oneC9ORF72 antisense transcript (herein “C9ORF72 antisense foci”). Incertain embodiments, C9ORF72 antisense foci comprise C9ORF72 antisensetranscripts comprising any of the following hexanucleotide repeats:GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC. In certain embodiments, C9ORF72foci comprise both C9ORF72 sense transcripts and C9ORF72 antisensetranscripts.

“C9ORF72 hexanucleotide repeat expansion associated disease” means anydisease associated with a C9ORF72 nucleic acid containing ahexanucleotide repeat expansion. In certain embodiments, thehexanucleotide repeat expansion may comprise any of the followinghexanucleotide repeats: GGGGCC, GGGGGG, GGGGGC, GGGGCG, GGCCCC, CCCCCC,GCCCCC, and/or CGCCCC. In certain embodiments, the hexanucleotide repeatis repeated at least 30 times, more than 30 times, more than 100 times,more than 500 times, or more than 1000 times. Such diseases may includea neurodegenerative disease. Such neurodegenerative diseases may includeALS and FTD.

“C9ORF72 nucleic acid” means any nucleic acid derived from the C9ORF72locus, regardless of which DNA strand the C9ORF72 nucleic acid isderived from. In certain embodiments, a C9ORF72 nucleic acid includes aDNA sequence encoding C9ORF72, an RNA sequence transcribed from DNAencoding C9ORF72 including genomic DNA comprising introns and exons(i.e., pre-mRNA), and an mRNA sequence encoding C9ORF72. “C9ORF72 mRNA”means an mRNA encoding a C9ORF72 protein. In certain embodiments, aC9ORF72 nucleic acid includes transcripts produced from the codingstrand of the C9ORF72 gene. C9ORF72 sense transcripts are examples ofC9ORF72 nucleic acids. In certain embodiments, a C9ORF72 nucleic acidincludes transcripts produced from the non-coding strand of the C9ORF72gene. C9ORF72 antisense transcripts are examples of C9ORF72 nucleicacids.

“C9ORF72 pathogenic associated mRNA variant” means the C9ORF72 mRNAvariant processed from a C9ORF72 pre-mRNA variant containing thehexanucleotide repeat. A C9ORF72 pre-mRNA contains the hexanucleotiderepeat when transcription of the pre-mRNA begins in the region from thestart site of exon 1A to the start site of exon 1B, e.g., nucleotides1107 to 1520 of the genomic sequence (SEQ ID NO: 2, the complement ofGENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000to 27565000). In certain embodiments, the level of a C9ORF72 pathogenicassociated mRNA variant is measured to determine the level of a C9ORF72pre-mRNA containing the hexanucleotide repeat in a sample.

“C9ORF72 transcript” means an RNA transcribed from C9ORF72. In certainembodiments, a C9ORF72 transcript is a C9ORF72 sense transcript. Incertain embodiments, a C9ORF72 transcript is a C9ORF72 antisensetranscript.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“cEt” or “constrained ethyl” means a bicyclic nucleoside having a sugarmoiety comprising a bridge connecting the 4′-carbon and the 2′-carbon,wherein the bridge has the formula: 4′-CH(CH₃)—O-2′.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleosidecomprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleosides is chemically distinct from a region having nucleosideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions, each position having a pluralityof subunits.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses parallel or sequentialadministration.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

“Comprise,” “comprises,” and “comprising” will be understood to implythe inclusion of a stated step or element or group of steps or elementsbut not the exclusion of any other step or element or group of steps orelements.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Designing” or “designed to” refer to the process of designing anoligomeric compound that specifically hybridizes with a selected nucleicacid molecule.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, in drugs that are injected, the diluent may be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections may be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses may be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” in the context of modulating an activity or oftreating or preventing a condition means the administration of thatamount of pharmaceutical agent to a subject in need of such modulation,treatment, or prophylaxis, either in a single dose or as part of aseries, that is effective for modulation of that effect, or fortreatment or prophylaxis or improvement of that condition. The effectiveamount may vary among individuals depending on the health and physicalcondition of the individual to be treated, the taxonomic group of theindividuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's codedinformation, regardless of which DNA strand the coded information isderived from, is converted into structures present and operating in acell. Such structures include, but are not limited to the products oftranscription and translation, including RAN translation.

“Fully complementary” or “100% complementary” means each nucleobase of afirst nucleic acid has a complementary nucleobase in a second nucleicacid. In certain embodiments, a first nucleic acid is an antisensecompound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap” and theexternal regions may be referred to as the “wings.”

“Gap-narrowed” means a chimeric antisense compound having a gap segmentof 9 or fewer contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from 1 to 6nucleosides.

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from 1 to 6nucleosides.

“Hexanucleotide repeat expansion” means a series of six bases (forexample, GGGGCC, GGGGGG, GGGGGC, GGGGCG, GGCCCC, CCCCCC, GCCCCC, and/orCGCCCC) repeated at least twice. In certain embodiments, thehexanucleotide repeat expansion may be located in intron 1 of a C9ORF72nucleic acid. In certain embodiments, the hexanucleotide repeat may betranscribed in the antisense direction from the C9ORF72 gene. In certainembodiments, a pathogenic hexanucleotide repeat expansion includes morethan 30, more than 100, more than 500, or more than 1000 repeats ofGGGGCC, GGGGGG, GGGGGC, GGGGCG, GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC ina C9ORF72 nucleic acid and is associated with disease. In certainembodiments, the repeats are consecutive. In certain embodiments, therepeats are interrupted by 1 or more nucleobases. In certainembodiments, a wild-type hexanucleotide repeat expansion includes 30 orfewer repeats of GGGGCC, GGGGGG, GGGGGC, GGGGCG, GGCCCC, CCCCCC, GCCCCC,and/or CGCCCC in a C9ORF72 nucleic acid. In certain embodiments, therepeats are consecutive. In certain embodiments, the repeats areinterrupted by 1 or more nucleobases.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude, but are not limited to, an antisense compound and a targetnucleic acid. In certain embodiments, complementary nucleic acidmolecules include, but are not limited to, an antisense oligonucleotideand a nucleic acid target.

“Hypoxanthine” (Hyp) also 6-Oxypurine is a purine derivative. In certainembodiments, a hypoxanthine (Hyp) may be used in place of a guanine (G)nucleobase to break up a series of 4 or more guanosines in a row(“G-quartet”). Hypoxanthine is a modified nucleobase.

“Identifying an animal having a C9ORF72 associated disease” meansidentifying an animal having been diagnosed with a C9ORF72 associateddisease or predisposed to develop a C9ORF72 associated disease.Individuals predisposed to develop a C9ORF72 associated disease includethose having one or more risk factors for developing a C9ORF72associated disease, including, having a personal or family history orgenetic predisposition of one or more C9ORF72 associated diseases. Incertain embodiments, the C9ORF72 associated disease is a C9ORF72hexanucleotide repeat expansion associated disease. Such identificationmay be accomplished by any method including evaluating an individual'smedical history and standard clinical tests or assessments, such asgenetic testing.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Inhibiting expression of a C9ORF72 antisense transcript” means reducingthe level or expression of a C9ORF72 antisense transcript and/or itsexpression products (e.g., RAN translation products). In certainembodiments, C9ORF72 antisense transcripts are inhibited in the presenceof an antisense compound targeting a C9ORF72 antisense transcript,including an antisense oligonucleotide targeting a C9ORF72 antisensetranscript, as compared to expression of C9ORF72 antisense transcriptlevels in the absence of a C9ORF72 antisense compound, such as anantisense oligonucleotide.

“Inhibiting expression of a C9ORF72 sense transcript” means reducing thelevel or expression of a C9ORF72 sense transcript and/or its expressionproducts (e.g., a C9ORF72 mRNA and/or protein). In certain embodiments,C9ORF72 sense transcripts are inhibited in the presence of an antisensecompound targeting a C9ORF72 sense transcript, including an antisenseoligonucleotide targeting a C9ORF72 sense transcript, as compared toexpression of C9ORF72 sense transcript levels in the absence of aC9ORF72 antisense compound, such as an antisense oligonucleotide.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity and does not necessarily indicatea total elimination of expression or activity.

“Inosine” (I) or 9-β-D-Ribosylhypoxanthine means a nucleoside thatcontains a hypoxanthine nucleobase.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Linked nucleosides” means adjacent nucleosides linked together by aninternucleoside linkage.

“Locked nucleic acid” or “LNA” or “LNA nucleosides” means nucleic acidmonomers having a bridge connecting two carbon atoms between the 4′ and2′position of the nucleoside sugar unit, thereby forming a bicyclicsugar. Examples of such bicyclic sugar include, but are not limited toA) α-L-Methyleneoxy (4′-CH₂—O-2′) LNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) LNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) LNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH₂—N(R)—O-2′) LNA, asdepicted below.

As used herein, LNA compounds include, but are not limited to, compoundshaving at least one bridge between the 4′ and the 2′ position of thesugar wherein each of the bridges independently comprises 1 or from 2 to4 linked groups independently selected from —[C(R₁)(R₂)]_(n)—,—C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—,—S(═O)_(x)— and —N(R₁)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4;each R₁ and R₂ is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, a heterocycle radical, a substitutedheterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclicradical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁),or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl or a protecting group.

Examples of 4′-2′ bridging groups encompassed within the definition ofLNA include, but are not limited to one of formulae: —[C(R₁)(R₂)]_(n)—,—[C(R₁)(R₂)]_(n)—O—, —C(R₁R₂)—N(R₁)—O— or —C(R₁R₂)—O—N(R₁)—.Furthermore, other bridging groups encompassed with the definition ofLNA are 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′,4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′-bridges, whereineach R₁ and R₂ is, independently, H, a protecting group or C₁-C₁₂ alkyl.

Also included within the definition of LNA according to the inventionare LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring isconnected to the 4′ carbon atom of the sugar ring, thereby forming amethyleneoxy (4′-CH₂—O-2′) bridge to form the bicyclic sugar moiety. Thebridge can also be a methylene (—CH₂—) group connecting the 2′ oxygenatom and the 4′ carbon atom, for which the term methyleneoxy(4′-CH₂—O-2′) LNA is used. Furthermore; in the case of the bicylic sugarmoiety having an ethylene bridging group in this position, the termethyleneoxy (4′ CH₂CH₂—O-2′) LNA is used. α-L-methyleneoxy(4′-CH₂—O-2′), an isomer of methyleneoxy (4′-CH₂—O-2′) LNA is alsoencompassed within the definition of LNA, as used herein.

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e., aphosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine,guanine, thymidine, or uracil. Hypoxanthine (Hyp) is a modifiednucleobase. An “unmodified nucleobase” means the purine bases adenine(A) and guanine (G); the pyrimidine bases thymine (T), cytosine (C), anduracil (U).

“Modified nucleoside” means a nucleoside having, independently, amodified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, and/or modifiednucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified internucleoside linkage, modified sugar, and/or modifiednucleobase.

“Modified sugar” means substitution and/or any change from a naturalsugar moiety.

“Monomer” means a single unit of an oligomer. Monomers include, but arenot limited to, nucleosides and nucleotides, whether naturally occurringor modified.

“Motif” means the pattern of unmodified and modified nucleoside in anantisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA(2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that donot form hydrogen bonds with one another or otherwise supporthybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes, but is not limited to, ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids,double-stranded nucleic acids, small interfering ribonucleic acids(siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable ofbase pairing with another nucleobase. For example, in DNA, adenine (A)is complementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). Hypoxanthine (Hyp) binds with adenine,thymine, or cytosine with a preference for binding with cytosine. Incertain embodiments, complementary nucleobase refers to a nucleobase ofan antisense compound that is capable of base pairing with a nucleobaseof its target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo, or tricyclo sugar mimetics, e.g., non furanose sugar units.Nucleotide mimetic includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage). Sugar surrogate overlaps with the slightly broader termnucleoside mimetic but is intended to indicate replacement of the sugarunit (furanose ring) only. The tetrahydropyranyl rings provided hereinare illustrative of an example of a sugar surrogate wherein the furanosesugar group has been replaced with a tetrahydropyranyl ring system.“Mimetic” refers to groups that are substituted for a sugar, anucleobase, and/or internucleoside linkage. Generally, a mimetic is usedin place of the sugar or sugar-internucleoside linkage combination, andthe nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Off-target effect” refers to an unwanted or deleterious biologicaleffect associated with modulation of RNA or protein expression of a geneother than the intended target nucleic acid.

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection(e.g., bolus injection) or infusion. Parenteral administration includessubcutaneous administration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g., intrathecal orintracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Without limitation, as used herein, peptide refers topolypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeuticbenefit when administered to an individual. In certain embodiments, anantisense oligonucleotide targeted to C9ORF72 sense transcript is apharmaceutical agent. In certain embodiments, an antisenseoligonucleotide targeted to C9ORF72 antisense transcript is apharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to as subject. For example, a pharmaceutical compositionmay comprise an antisense oligonucleotide and a sterile aqueoussolution.

“Pharmaceutically acceptable derivative” encompasses pharmaceuticallyacceptable salts, conjugates, prodrugs or isomers of the compoundsdescribed herein.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked)nucleobases of a nucleic acid. In certain embodiments, a portion is adefined number of contiguous nucleobases of a target nucleic acid. Incertain embodiments, a portion is a defined number of contiguousnucleobases of an antisense compound.

“Prevent” or “preventing” refers to delaying or forestalling the onsetor development of a disease, disorder, or condition for a period of timefrom minutes to days, weeks to months, or indefinitely.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Prophylactically effective amount” refers to an amount of apharmaceutical agent that provides a prophylactic or preventativebenefit to an animal.

“Region” is defined as a portion of the target nucleic acid having atleast one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ positionof the sugar portion of the nucleotide. Ribonucleotides may be modifiedwith any of a variety of substituents.

“Salts” mean a physiologically and pharmaceutically acceptable salts ofantisense compounds, i.e., salts that retain the desired biologicalactivity of the parent oligonucleotide and do not impart undesiredtoxicological effects thereto.

“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid.

“Shortened” or “truncated” versions of antisense oligonucleotides taughtherein have one, two or more nucleosides deleted.

“Side effects” means physiological responses attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Sites,” as used herein, are defined as unique nucleobase positionswithin a target nucleic acid.

“Slows progression” means decrease in the development of the disease.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity between an antisenseoligonucleotide and a target nucleic acid to induce a desired effect,while exhibiting minimal or no effects on non-target nucleic acids underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays and therapeutictreatments.

“Stringent hybridization conditions” or “stringent conditions” refer toconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby antisense compounds.

“Target region” means a portion of a target nucleic acid to which one ormore antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an individual.

“Treat” or “treating” or “treatment” means administering a compositionto effect an alteration or improvement of a disease or condition.

“Unmodified nucleobases” means the purine bases adenine (A) and guanine(G), and the pyrimidine bases (T), cytosine (C), and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

“Wing segment” means a plurality of nucleosides modified to impart to anoligonucleotide properties such as enhanced inhibitory activity,increased binding affinity for a target nucleic acid, or resistance todegradation by in vivo nucleases.

Certain Embodiments

Provided herein are compounds comprising a C9ORF72 antisense transcriptspecific inhibitor.

In certain embodiments, the C9ORF72 antisense transcript specificinhibitor is an antisense compound.

In certain embodiments, the C9ORF72 antisense transcript specificantisense compound is an antisense oligonucleotide.

In certain embodiments, the antisense oligonucleotide consists of 12-30linked nucleosides.

In certain embodiments, the antisense oligonucleotide consists of 16-25linked nucleosides.

In certain embodiments, the antisense oligonucleotide consists of 18-22linked nucleosides

In certain embodiments, the antisense oligonucleotide has a nucleobasesequence that is at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to aC9ORF72 antisense transcript.

In certain embodiments, the antisense oligonucleotide has a nucleobasesequence that is at least 90% complementary to a C9ORF72 antisensetranscript.

In certain embodiments, the antisense oligonucleotide has a nucleobasesequence that is at least 95% complementary to a C9ORF72 antisensetranscript.

In certain embodiments, the antisense oligonucleotide has a nucleobasesequence that is 100% complementary to a C9ORF72 antisense transcript.

In certain embodiments, the C9ORF72 antisense transcript has thenucleobase sequence of SEQ ID NO: 13.

In certain embodiments, the antisense oligonucleotide has a nucleobasesequence comprising at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, or 20 contiguous nucleobases of a sequenceselected from among SEQ ID NO: 30-84.

In certain embodiments, the antisense oligonucleotide is a modifiedantisense oligonucleotide.

In certain embodiments, the modified antisense oligonucleotide comprisesat least one modified internucleoside linkage.

In certain embodiments, each modified internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments, the at least one modified internucleosidelinkage is a phosphorothioate internucleoside linkage.

In certain embodiments, the modified antisense oligonucleotide comprisesat least one phosphodiester internucleoside linkage.

In certain embodiments, at least one modified nucleobase is ahypoxanthine.

In certain embodiments, at least one nucleoside of the modifiedantisense oligonucleotide is an inosine.

In certain embodiments, the at least one nucleoside of the modifiedantisense oligonucleotide comprises a modified nucleobase.

In certain embodiments, at least one modified nucleobase is a5-methylcytosine.

In certain embodiments, the at least one nucleoside of the modifiedantisense oligonucleotide comprises a modified sugar.

In certain embodiments, the at least one modified sugar is a bicyclicsugar.

In certain embodiments, the bicyclic sugar comprises a chemical bridgebetween the 2′ and 4′ position of the sugar, wherein the chemical bridgeis selected from: 4′-CH₂—O-2′; 4′-CH(CH₃)—O-2′; 4′-(CH₂)₂—O-2′; and4′-CH₂—N(R)—O-2′ wherein R is, independently, H, C₁-C₁₂ alkyl, or aprotecting group.

In certain embodiments, the at least one modified sugar comprises a2′-O-methoxyethyl group.

In certain embodiments, the antisense oligonucleotide is a gapmer.

In certain embodiments, the compound comprises at least one conjugate.

In certain embodiments, the C9ORF72 antisense transcript specificantisense compound consists of an antisense oligonucleotide.

Provided herein are pharmaceutical compositions comprising any compounddescribed herein and a pharmaceutically acceptable diluent or carrier.

Provided herein are pharmaceutical compositions comprising a C9ORF72antisense transcript specific inhibitor.

Provided herein are pharmaceutical compositions comprising a C9ORF72antisense transcript specific inhibitor and a C9ORF sense transcriptspecific inhibitor.

In certain embodiments, the C9ORF72 sense transcript specific inhibitoris a C9ORF72 sense transcript specific antisense compound.

In certain embodiments, the C9ORF72 antisense transcript specificinhibitor is a C9ORF72 antisense transcript specific antisense compound.

In certain embodiments, the C9ORF72 sense transcript specific antisensecompound is an antisense oligonucleotide.

In certain embodiments, the C9ORF72 antisense transcript specificantisense compound is an antisense oligonucleotide.

In certain embodiments, the C9ORF72 antisense transcript has thenucleobase sequence of SEQ ID NO: 13.

In certain embodiments, the C9ORF72 sense transcript has the nucleobasesequence of SEQ ID NO: 1-10.

Provided herein are uses of any compound described herein for themanufacture of a medicament for treating a neurodegenerative disease.

Provided herein are methods, comprising contacting a cell with anantisense oligonucleotide having a nucleobase sequence of any of SEQ IDNOs: 30-84.

Provided herein are methods, comprising contacting a cell with a C9ORF72antisense transcript specific inhibitor.

Provided herein are methods, comprising contacting a cell with a C9ORF72antisense transcript specific inhibitor and a C9ORF72 sense transcriptspecific inhibitor.

Provided herein are methods, comprising contacting a cell with a C9ORF72antisense transcript specific inhibitor; and thereby reducing the levelor expression of C9ORF72 antisense transcript in the cell.

Provided herein are methods, comprising contacting a cell with a C9ORF72antisense transcript specific inhibitor and a C9ORF72 sense transcriptspecific inhibitor; and thereby reducing the level or expression of bothC9ORF72 antisense transcript and C9ORF72 sense transcript in the cell.

In certain embodiments, the C9ORF72 antisense specific inhibitor is anantisense compound.

In certain embodiments, the C9ORF72 antisense transcript specificinhibitor is an antisense compound.

In certain embodiments, the cell is in vitro.

In certain embodiments, the cell is in an animal.

Provided herein are methods, comprising administering to an animal inneed thereof a therapeutically effective amount of a C9ORF72 antisensetranscript specific inhibitor.

In certain embodiments the amount is effective to reduce the level orexpression of the C9ORF72 antisense transcript.

Provided herein are methods, comprising co-administering to an animal inneed thereof a therapeutically effective amount of a C9ORF72 antisensetranscript inhibitor and a therapeutically effective amount of a C9ORF72sense transcript inhibitor.

In certain embodiments the therapeutically effective amount is effectiveto reduce the level or expression of the C9ORF72 antisense transcriptand the C9ORF72 sense transcript.

In certain embodiments, wherein the C9ORF72 antisense transcriptinhibitor is a C9ORF72 antisense transcript specific antisense compound.

In certain embodiments, the C9ORF72 sense transcript inhibitor is aC9ORF72 sense transcript specific antisense compound.

Provided herein are methods, comprising:

identifying an animal having a C9ORF72 associated disease; and

administering to the animal a therapeutically effective amount of aC9ORF72 antisense transcript specific inhibitor.

In certain embodiments the amount is effective to reduce the level orexpression of the C9OR72 antisense transcript.

Provided herein are methods, comprising:

identifying an animal having a C9ORF72 associated disease; and

coadministering to the animal a therapeutically effective amount of aC9ORF72 antisense transcript specific inhibitor and a therapeuticallyeffective amount of a C9ORF72 sense transcript inhibitor.

In certain embodiments the amount is effective to reduce the level orexpression of the C9ORF72 antisense transcript and the C9ORF72 sensetranscript.

In certain embodiments the C9ORF72 antisense transcript specificinhibitor is a C9ORF72 antisense transcript specific antisense compound.

In certain embodiments the C9ORF72 sense transcript inhibitor is aC9ORF72 sense transcript specific antisense compound.

In certain embodiments the C9ORF72 antisense transcript specificantisense compound is at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% complementary toa C9ORF72 anti sense transcript.

In certain embodiments the C9ORF72 sense transcript specific antisensecompound is at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to aC9ORF72 sense transcript.

In certain embodiments the C9ORF72 antisense transcript is SEQ ID NO:13.

In certain embodiments the C9ORF72 sense transcript is any of SEQ ID NO:1-10.

In certain embodiments the C9ORF72 associated disease is a C9ORF72hexanucleotide repeat expansion associated disease.

In certain embodiments the C9ORF72 associated disease or C9ORF72hexanucleotide repeat expansion associated disease is amyotrophiclateral sclerosis (ALS), frontotemporal dementia (FTD), corticobasaldegeneration syndrome (CBD), atypical Parkinsonian syndrome, andolivopontocerebellar degeneration (OPCD).

In certain embodiments the amyotrophic lateral sclerosis (ALS) isfamilial ALS or sporadic ALS.

In certain embodiments the contacting or administering reduces C9ORF72antisense transcript associated RAN translation products.

In certain embodiments the C9ORF72 antisense transcript associated RANtranslation products are any of poly-(proline-alanine),poly-(proline-arginine), and poly-(proline-glycine).

In certain embodiments the administering and coadminstering isparenteral administration.

In certain embodiments the parental administration is any of injectionor infusion.

In certain embodiments the parenteral administration is any ofintrathecal administration or intracerebroventricular administration.

In certain embodiments at least one symptom of a C9ORF72 associateddisease or a C9ORF72 hexanucleotide repeat expansion associated diseaseis slowed, ameliorated, or prevented.

In certain embodiments the at least one symptom is any of motorfunction, respiration, muscle weakness, fasciculation and cramping ofmuscles, difficulty in projecting the voice, shortness of breath,difficulty in breathing and swallowing, inappropriate social behavior,lack of empathy, distractibility, changes in food preferences,agitation, blunted emotions, neglect of personal hygiene, repetitive orcompulsive behavior, and decreased energy and motivation.

In certain embodiments the C9ORF72 antisense transcript specificantisense compound is an antisense oligonucleotide.

In certain embodiments the C9ORF72 sense transcript specific antisensecompound is an antisense oligonucleotide.

In certain embodiments the antisense oligonucleotide is a modifiedantisense oligonucleotide.

In certain embodiments at least one internucleoside linkage of theantisense oligonucleotide is a modified internucleoside linkage.

In certain embodiments at least one modified internucleoside linkage isa phosphorothioate internucleoside linkage.

In certain embodiments each modified internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments at least one nucleoside of the modified antisenseoligonucleotide comprises a modified nucleobase.

In certain embodiments the modified nucleobase is a 5-methylcytosine.

In certain embodiments at least one nucleoside of the modified antisenseoligonucleotide comprises a modified sugar.

In certain embodiments at least one modified sugar is a bicyclic sugar.

In certain embodiments the bicyclic sugar comprises a chemical bridgebetween the 2′ and 4′ position of the sugar, wherein the chemical bridgeis selected from: 4′-CH₂—O-2′; 4′-CH(CH₃)—O-2′; 4′-(CH₂)₂—O-2′; and4′-CH₂—N(R)—O-2′ wherein R is, independently, H, C₁-C₁₂ alkyl, or aprotecting group.

In certain embodiments at least one modified sugar comprises a2′-O-methoxyethyl group.

In certain embodiments the antisense oligonucleotide is a gapmer.

The present disclosure provides the following non-limiting numberedembodiments: Embodiment 1. A compound comprising a modifiedoligonucleotide consisting of 12-30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases of any of the nucleobases sequences of SEQ ID NOs: 30-99.

Embodiment 2. The compound of embodiment 1, wherein the modifiedoligonucleotide has a nucleobase sequence that is at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% complementary to a C9ORF72 antisense transcript.

Embodiment 3. The compound of embodiment 2, wherein the C9ORF72antisense transcript has the nucleobase sequence of SEQ ID NO: 13.

Embodiment 4. The compound of any of embodiments 1-3, wherein themodified oligonucleotide is a single-stranded modified oligonucleotide.

Embodiment 5. The compound of any of embodiments 1-4, wherein themodified oligonucleotide comprises at least one modified internucleosidelinkage.

Embodiment 6. The compound of any of embodiment 5, wherein the at leastone modified internucleoside linkage is a phosphorothioateinternucleoside linkage.

Embodiment 7. The compound of embodiments 5 or 6, wherein the modifiedoligonucleotide comprises at least one phosphodiester linkage.

Embodiment 8. The compound of embodiment 6, wherein each internucleosidelinkage is a phosphorothioate internucleoside linkage.

Embodiment 9. The compound of any of embodiments 1-8, wherein at leastone nucleoside of the modified oligonucleotide comprises a modifiednucleobase.

Embodiment 10. The compound of embodiment 9, wherein the modifiednucleobase is a 5 methylcytosine.

Embodiment 11. The compound of any of embodiments 1-10, wherein at leastone nucleoside of the modified oligonucleotide comprises a modifiedsugar.

Embodiment 12. The compound of embodiment 11, wherein each nucleoside ofthe modified oligonucleotide comprises a modified sugar.

Embodiment 13. The compound of embodiments 11 or 12, wherein themodified sugar is a bicyclic sugar.

Embodiment 14. The compound of embodiment 13, wherein the bicyclic sugarcomprises a chemical bridge between the 4′ and 2′ positions of thesugar, wherein the chemical bridge is selected from: 4′-CH(R)—O-2′ and4′-(CH₂)₂—O-2′, wherein R is independently selected from H, C₁-C₆ alkyl,and C₁-C₆ alkoxy.

Embodiment 15. The compound of embodiment 14, wherein the chemicalbridge is 4′-CH(R)-0-2′ and wherein R is methyl.

Embodiment 16. The compound of embodiment 14, wherein the chemicalbridge is 4′-CH(R)-0-2′ and wherein R is H.

Embodiment 17. The compound of embodiment 14, wherein the chemicalbridge is 4′-CH(R)-0-2′ and wherein R is —CH₂—O—CH₃.

Embodiment 18. The compound of embodiments 11 or 12, wherein themodified sugar comprises a 2′-O-methoxyethyl group.

Embodiment 19. The compound of any of embodiments 1-11 and 13-18,wherein the modified oligonucleotide is a gapmer.

Embodiment 20. The compound of embodiment 19, wherein the gapmer isselected from a 5-10-5 MOE gapmer, a 5-8-5 MOE gapmer, or a 4-8-4 MOEgapmer.

Embodiment 21. A pharmaceutical composition comprising the compound ofany preceding embodiment or salt thereof and at least one of apharmaceutically acceptable carrier or diluent.

Embodiment 22. The pharmaceutical composition of embodiment 21 furthercomprising a C9ORF72 sense transcript specific inhibitor.

Embodiment 23. The pharmaceutical composition of embodiment 22, whereinthe C9ORF72 sense transcript specific inhibitor is any of a nucleicacid, aptamer, antibody, peptide, or small molecule.

Embodiment 24. The pharmaceutical composition of embodiment 23, whereinthe nucleic acid is a single-stranded nucleic acids or a double-strandednucleic acid.

Embodiment 25. The pharmaceutical composition of embodiment 23, whereinthe nucleic acid is a siRNA.

Embodiment 26. The pharmaceutical composition of embodiment 22, whereinthe C9ORF72 sense transcript inhibitor is an antisense compound.

Embodiment 27. The pharmaceutical composition of embodiment 26, whereinthe antisense compound is an antisense oligonucleotide.

Embodiment 28. The pharmaceutical composition of embodiment 26, whereinthe antisense compound is a modified oligonucleotide.

Embodiment 29. The pharmaceutical composition of embodiment 28, whereinthe modified oligonucleotide is single-stranded.

Embodiment 30. The pharmaceutical composition of embodiments 28 or 29,wherein the modified oligonucleotide has a nucleobase sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% complementary to a C9ORF72 sensetranscript.

Embodiment 31. The pharmaceutical composition of embodiment 30 whereinthe C9ORF72 sense transcript has the nucleobase sequence of SEQ ID NO:1-10.

Embodiment 32. Use of the compound or composition of any precedingembodiment for the manufacture of a medicament for treating aneurodegenerative disease.

Embodiment 33. A method comprising administering to an animal thecompound or composition of any preceding embodiment.

Embodiment 34. The method of embodiment 33, wherein the compoundprevents, treats, ameliorates, or slows progression of at least onesymptom of a C9ORF72 associated disease.

Embodiment 35. The method of embodiment 34, wherein the at least onesymptom is selected from among impaired motor function, difficulty withrespiration, muscle weakness, fasciculation and cramping of muscles,difficulty in projecting the voice, shortness of breath, difficulty inbreathing and swallowing, inappropriate social behavior, lack ofempathy, distractibility, changes in food preference, agitation, bluntedemotions, neglect of personal hygiene, repetitive or compulsivebehavior, and decreased energy and motivation.

Embodiment 36. The method of embodiment 34, wherein the C9ORF72associated disease is amyotrophic lateral sclerosis (ALS),frontotemporal dementia (FTD), corticobasal degeneration syndrome (CBD),atypical Parkinsonian syndrome, or olivopontocerebellar degeneration(OPCD).

Embodiment 37. The method of embodiment 36, wherein the amyotrophiclateral sclerosis (ALS) is familial ALS.

Embodiment 38. The method of embodiment 36, wherein the amyotrophiclateral sclerosis (ALS) is sporadic ALS.

Embodiment 39. The method of any of embodiments 33-38, wherein theadministering reduces C9ORF72 antisense transcript associated RANtranslation products.

Embodiment 40. The method of embodiment 39, wherein the C9ORF72antisense transcript associated RAN translation products are any ofpoly-(proline-alanine), poly-(proline-arginine), andpoly-(proline-glycine).

Embodiment 41. The method of any of embodiments 33-40, wherein theadministering reduces C9ORF72 antisense foci.

Embodiment 42. The method of any of embodiments 33-41, wherein theadministering reduces C9ORF72 sense foci.

Embodiment 43. The method of any of embodiments 33-42, wherein theadministering is parenteral administration.

Embodiment 44. The method of embodiment 43, wherein the parenteraladministration is any of injection or infusion.

Embodiment 45. The method of embodiment 43, wherein the parenteraladministration is directly into the central nervous system (CNS).

Embodiment 46. The method of any of embodiments 43-45, wherein theparenteral administration is any of intrathecal administration orintracerebroventricular administration.

Embodiment 47. The compound of embodiment 11, wherein the modifiedoligonucleotide comprises sugar residues in any of the followingpatterns: eeedeeeeedeeeeedeeee or eeeeedeeeeedeeeeee, wherein,

-   -   e=a 2′-O-methoxyethylribose modified sugar, and    -   d=a 2′-deoxyribose sugar.

Embodiment 48. The compound of embodiment 5, wherein the modifiedoligonucleotide comprises internucleoside linkages in any of thefollowing patterns: soooossssssssssooss, sooosssssssssooss,ssososssososssososs, or ssssososssosossss, wherein,

-   -   s=a phosphorothioate linkage, and    -   o=a phosphodiester linkage.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound may be “antisense” to a target nucleic acid, meaningthat is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a C9ORF72nucleic acid is 12 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits. In certainembodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18to 24, 19 to 22, or 20 linked subunits. In certain embodiments, theantisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a rangedefined by any two of the above values. In some embodiments theantisense compound is an antisense oligonucleotide, and the linkedsubunits are nucleosides.

In certain embodiments antisense oligonucleotides targeted to a C9ORF72nucleic acid may be shortened or truncated. For example, a singlesubunit may be deleted from the 5′ end (5′ truncation), or alternativelyfrom the 3′ end (3′ truncation). A shortened or truncated antisensecompound targeted to a C9ORF72 nucleic acid may have two subunitsdeleted from the 5′ end, or alternatively may have two subunits deletedfrom the 3′ end, of the antisense compound. Alternatively, the deletednucleosides may be dispersed throughout the antisense compound, forexample, in an antisense compound having one nucleoside deleted from the5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisensecompound, the additional subunit may be located at the 5′ or 3′ end ofthe antisense compound. When two or more additional subunits arepresent, the added subunits may be adjacent to each other, for example,in an antisense compound having two subunits added to the 5′ end (5′addition), or alternatively to the 3′ end (3′ addition), of theantisense compound. Alternatively, the added subunits may be dispersedthroughout the antisense compound, for example, in an antisense compoundhaving one subunit added to the 5′ end and one subunit added to the 3′end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358,1988) tested a series oftandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a C9ORF72nucleic acid have chemically modified subunits arranged in patterns, ormotifs, to confer to the antisense compounds properties such as enhancedinhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNaseH cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anantisense oligonucleotide having a gapmer motif, the gap segmentgenerally serves as the substrate for endonuclease cleavage, while thewing segments comprise modified nucleosides. In certain embodiments, theregions of a gapmer are differentiated by the types of sugar moietiescomprising each distinct region. The types of sugar moieties that areused to differentiate the regions of a gapmer may in some embodimentsinclude β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modifiednucleosides (such 2′-modified nucleosides may include 2′-MOE, and2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (suchbicyclic sugar modified nucleosides may include those having a4′-(CH₂)n-O-2′ bridge, where n=1 or n=2 and 4′-CH₂—O—CH₂-2′).Preferably, each distinct region comprises uniform sugar moieties. Thewing-gap-wing motif is frequently described as “X-Y-Z”, where “X”represents the length of the 5′ wing region, “Y” represents the lengthof the gap region, and “Z” represents the length of the 3′ wing region.As used herein, a gapmer described as “X-Y-Z” has a configuration suchthat the gap segment is positioned immediately adjacent to each of the5′ wing segment and the 3′ wing segment. Thus, no interveningnucleotides exist between the 5′ wing segment and gap segment, or thegap segment and the 3′ wing segment. Any of the antisense compoundsdescribed herein can have a gapmer motif. In some embodiments, X and Zare the same, in other embodiments they are different. In a preferredembodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30 or more nucleotides. Thus, gapmers described herein include, butare not limited to, for example 5-10-5, 5-10-4, 4-10-4, 4-10-3, 3-10-3,2-10-2, 5-9-5, 5-9-4, 4-9-5, 5-8-5, 5-8-4, 4-8-5, 5-7-5, 4-7-5, 5-7-4,or 4-7-4.

In certain embodiments, the antisense compound has a “wingmer” motif,having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Zconfiguration as described above for the gapmer configuration. Thus,wingmer configurations described herein include, but are not limited to,for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10,8-2, 2-13, 5-13, 5-8, or 6-8.

In certain embodiments, an antisense compound targeted to a C9ORF72nucleic acid has a gap-narrowed motif. In certain embodiments, agap-narrowed antisense oligonucleotide targeted to a C9ORF72 nucleicacid has a gap segment of 9, 8, 7, or 6 2′-deoxynucleotides positionedimmediately adjacent to and between wing segments of 5, 4, 3, 2, or 1chemically modified nucleosides. In certain embodiments, the chemicalmodification comprises a bicyclic sugar. In certain embodiments, thebicyclic sugar comprises a 4′ to 2′ bridge selected from among:4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or 2; and 4′-CH₂—O—CH₂-2′. Incertain embodiments, the bicyclic sugar is comprises a 4′-CH(CH₃)—O-2′bridge. In certain embodiments, the chemical modification comprises anon-bicyclic 2′-modified sugar moiety. In certain embodiments, thenon-bicyclic 2′-modified sugar moiety comprises a 2′-O-methylethyl groupor a 2′-O-methyl group.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode C9ORF72 include, without limitation,the following: the complement of GENBANK Accession No. NM 001256054.1(incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT008413.18 truncated from nucleobase 27535000 to 27565000 (incorporatedherein as SEQ ID NO: 2), GENBANK Accession No. BQ068108.1 (incorporatedherein as SEQ ID NO: 3), GENBANK Accession No. NM 018325.3 (incorporatedherein as SEQ ID NO: 4), GENBANK Accession No. DN993522.1 (incorporatedherein as SEQ ID NO: 5), GENBANK Accession No. NM 145005.5 (incorporatedherein as SEQ ID NO: 6), GENBANK Accession No. DB079375.1 (incorporatedherein as SEQ ID NO: 7), GENBANK Accession No. BU194591.1 (incorporatedherein as SEQ ID NO: 8), Sequence Identifier 4141_014_A (incorporatedherein as SEQ ID NO: 9), and Sequence Identifier 4008_73_A (incorporatedherein as SEQ ID NO: 10).

Nucleotide sequences that encode the C9ORF72 antisense transcriptinclude, without limitation, the following: SEQ ID NO: 13. The sequenceof SEQ ID NO: 13 is complementary to nucleotides 1159 to 1929 of SEQ IDNO: 2 (the complement of GENBANK Accession No. NT 008413.18 truncatedfrom nucleotides 27535000 to 27565000) except that SEQ ID NO: 13 has twomore hexanucleotide repeats than SEQ ID NO: 2. The sequence of thehexanucleotide repeat is GGCCCC in SEQ ID NO: 13 and GGGGCC in SEQ IDNO: 2. Thus, SEQ ID NO: 13 is 12 nucleotides longer than nucleotides1159 to 1929 of SEQ ID NO: 2, to which it is complementary.

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor C9ORF72 can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region may encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the same target region.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceeding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin a target region. In certain embodiments, reductions in C9ORF72mRNA levels are indicative of inhibition of C9ORF72 expression.Reductions in levels of a C9ORF72 protein are also indicative ofinhibition of target mRNA expression. Reduction in the presence ofexpanded C9ORF72 RNA foci are indicative of inhibition of C9ORF72expression. Further, phenotypic changes are indicative of inhibition ofC9ORF72 expression. For example, improved motor function and respirationmay be indicative of inhibition of C9ORF72 expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a C9ORF72 nucleic acid. The most common mechanismof hybridization involves hydrogen bonding (e.g., Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the antisense compounds provided herein are specificallyhybridizable with a C9ORF72 nucleic acid.

Complementarily

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a C9ORF72 nucleicacid).

Non-complementary nucleobases between an antisense compound and aC9ORF72 nucleic acid may be tolerated provided that the antisensecompound remains able to specifically hybridize to a target nucleicacid. Moreover, an antisense compound may hybridize over one or moresegments of a C9ORF72 nucleic acid such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to a C9ORF72 nucleic acid, a target region, targetsegment, or specified portion thereof. Percent complementarity of anantisense compound with a target nucleic acid can be determined usingroutine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e., 100%complementary) to a target nucleic acid, or specified portion thereof.For example, an antisense compound may be fully complementary to aC9ORF72 nucleic acid, or a target region, or a target segment or targetsequence thereof. As used herein, “fully complementary” means eachnucleobase of an antisense compound is capable of precise base pairingwith the corresponding nucleobases of a target nucleic acid. Forexample, a 20 nucleobase antisense compound is fully complementary to atarget sequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound may or may not be fully complementary tothe target sequence, depending on whether the remaining 10 nucleobasesof the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e., linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no morethan 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a C9ORF72 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a C9ORF72 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 9 nucleobase portion of a target segment. Incertain embodiments, the antisense compounds are complementary to atleast a 10 nucleobase portion of a target segment. In certainembodiments, the antisense compounds, are complementary to at least an11 nucleobase portion of a target segment. In certain embodiments, theantisense compounds, are complementary to at least a 12 nucleobaseportion of a target segment. In certain embodiments, the antisensecompounds, are complementary to at least a 13 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds, arecomplementary to at least a 14 nucleobase portion of a target segment.In certain embodiments, the antisense compounds, are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least a 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a targetsegment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases may be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is comparedto an equal length portion of the target nucleic acid. In certainembodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleobase portion is compared to an equal lengthportion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide iscompared to an equal length portion of the target nucleic acid. Incertain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equallength portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to a C9ORF72nucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, the modified internucleoside linkages areinterspersed throughout the antisense compound. In certain embodiments,the modified internucleoside linkages are phosphorothioate linkages. Incertain embodiments, each internucleoside linkage of an antisensecompound is a phosphorothioate internucleoside linkage. In certainembodiments, the antisense compounds targeted to a C9ORF72 nucleic acidcomprise at least one phosphodiester linkage and at least onephosphorothioate linkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprisechemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂are each independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_(m))(R_(n)), where each R_(l), R_(m) andR_(n) is, independently, H or substituted or unsubstituted C1-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleic acids(BNAs) include without limitation nucleosides comprising a bridgebetween the 4′ and the 2′ ribosyl ring atoms. In certain embodiments,antisense compounds provided herein include one or more BNA nucleosideswherein the bridge comprises one of the formulas: 4′-(CH₂)—O-2′ (LNA);4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845,issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof seePCT/US2008/068922 published as WO/2009/006478, published Jan. 8, 2009);4′-CH₂—N(OCH₃)-2′ (and analogs thereof see PCT/US2008/064591 publishedas WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (seepublished U.S. Patent Application US2004-0171570, published Sep. 2,2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protectinggroup (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008);4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya et al., J. Org. Chem., 2009, 74,118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof seePCT/US2008/066154 published as WO 2008/154401, published on Dec. 8,2008).

Further bicyclic nucleosides have been reported in published literature(see for example: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26)8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372;Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braaschet al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol.Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci.U.S.A, 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4,455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org.Chem., 1998, 63, 10035-10039; U.S. Pat. Nos. 7,399,845; 7,053,207;7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S.Patent Publication Nos.: US2008-0039618; US2007-0287831; US2004-0171570;U.S. patent application Ser. Nos. 12/129,154; 61/099,844; 61/097,787;61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574;International applications WO 2007/134181; WO 2005/021570; WO2004/106356; and PCT International Applications Nos.: PCT/US2008/068922;PCT/US2008/066154; and PCT/US2008/064591). Each of the foregoingbicyclic nucleosides can be prepared having one or more stereochemicalsugar configurations including for example α-L-ribofuranose andβ-D-ribofuranose (see PCT international application PCT/DK98/00393,published on Mar. 25, 1999 as WO 99/14226).

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ carbon atoms of the pentofuranosyl sugarmoiety including without limitation, bridges comprising 1 or from 1 to 4linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; nis 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, aprotecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycleradical, substituted heterocycle radical, heteroaryl, substitutedheteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclicradical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H),substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a4′-(CH₂)—O-2′ bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4′to 2′ bridge wherein such bridges include without limitation,α-L-4′-(CH₂)—O-2′, β-D-4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′,4′-CH₂—N(R)—O-2′, 4′-CH(CH₃)—O-2′, 4′-CH₂—S-2′, 4′-CH₂—N(R)-2′,4′-CH₂—CH(CH₃)-2′, and 4′-(CH₂)₃-2′, wherein R is H, a protecting groupor C₁-C₁₂ alkyl.

In certain embodiment, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C2-C6 alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

-   -   Bx is a heterocyclic base moiety;    -   T_(a) and T_(b) are each, independently H, a hydroxyl protecting        group, a conjugate group, a reactive phosphorus group, a        phosphorus moiety or a covalent attachment to a support medium;    -   R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,        substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C2-C6        alkynyl;    -   each q_(a), q_(b), q_(c) and q_(d) is, independently, H,        halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,        substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆        alkynyl, C₁-C₆ alkoxyl, substituted C₁-C₆ alkoxyl, acyl,        substituted acyl, C₁-C₆ aminoalkyl or substituted C₁-C₆        aminoalkyl;

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of adenine, cytosine, guanine,5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a4′-CH₂—O-2′ bridge, along with their oligomerization, and nucleic acidrecognition properties have been described (Koshkin et al., Tetrahedron,1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has alsobeen described in WO 98/39352 and WO 99/14226.

Analogs of various bicyclic nucleosides that have 4′ to 2′ bridginggroups such as 4′-CH₂—O-2′ and 4′-CH₂—S-2′, have also been prepared(Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of oligodeoxyribonucleotide duplexes comprising bicyclicnucleosides for use as substrates for nucleic acid polymerases has alsobeen described (Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel conformationally restricted high-affinityoligonucleotide analog has been described in the art (Singh et al., J.Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJT, SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k),N₃, CN, C(═O)OJ_(j)J_(k), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Frier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy(4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to asconstrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H)methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA,and (K) vinyl BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protectinggroup, C₁-C₆ alkyl or C₁-C₆ alkoxy.

As used herein, the term “modified tetrahydropyran nucleoside” or“modified THP nucleoside” means a nucleoside having a six-memberedtetrahydropyran “sugar” substituted for the pentofuranosyl residue innormal nucleosides and can be referred to as a sugar surrogate. ModifiedTHP nucleosides include, but are not limited to, what is referred to inthe art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10,841-854) or fluoro HNA (F-HNA) having a tetrahydropyranyl ring system asillustrated below.

In certain embodiment, sugar surrogates are selected having the formula:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an oligomeric compound oroligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁,J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isother than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄,q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following formula:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenylnucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the cyclohexenyl nucleoside analog to an antisense compound orone of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an antisense compound and the otherof T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugarsubstituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known inthe art and are suitable as sugar surrogates that can be used to modifynucleosides for incorporation into oligomeric compounds as providedherein (see for example review article: Leumann, Christian J. Bioorg. &Med. Chem., 2002, 10, 841-854). Such ring systems can undergo variousadditional substitutions to further enhance their activity.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′- substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifiednucleosides comprise a 2′ MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃”, “2′-O-methyl” or “2′-methoxy”each refers to a nucleoside comprising a sugar comprising an —OCH₃ groupat the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

Methods for the preparations of modified sugars are well known to thoseskilled in the art. Some representative U.S. patents that teach thepreparation of such modified sugars include without limitation, U.S.:4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633;5,700,920; 5,792,847 and 6,600,032 and International ApplicationPCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 onDec. 22, 2005, and each of which is herein incorporated by reference inits entirety.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the(4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wingsof a gapmer motif.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active or inert substances for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

An antisense compound targeted to a C9ORF72 nucleic acid can be utilizedin pharmaceutical compositions by combining the antisense compound witha suitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to a C9ORF72 nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is PBS. In certain embodiments, the antisensecompound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof C9ORF72 nucleic acids can be tested in vitro in a variety of celltypes. Cell types used for such analyses are available from commercialvendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio,Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville,Md.) and are cultured according to the vendor's instructions usingcommercially available reagents (e.g. Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, and primary hepatocytes.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a LIPOFECTAMINEconcentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation.

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE. Antisense oligonucleotides are used at higherconcentrations ranging from 625 to 20,000 nM when transfected usingelectroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOLReagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a C9ORF72 nucleic acid can beassayed in a variety of ways known in the art. For example, targetnucleic acid levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or quantitative real-timePCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence DetectionSystem (PE-Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. Methods of quantitative real-time PCR arewell known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000instrument (PE Applied Biosystems) is used to measure RIBOGREENfluorescence.

Probes and primers are designed to hybridize to a C9ORF72 nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESSSoftware (Applied Biosystems, Foster City, Calif.).

Strand Specific Semi-Quantitative PCR Analysis of Target RNA Levels

Analysis of specific, low abundance target RNA strand levels may beaccomplished by reverse transcription, PCR, and gel densitometryanalysis using the Gel Logic 200 Imaging System and Kodak MI software(Kodak Scientific Imaging Systems, Rochester, N.Y., USA) according tomanufacturer's instructions.

RT-PCR reactions are carried out as taught in Ladd, P. D., et al, (HumanMolecular Genetics, 2007, 16, 3174-3187) and in Sopher, B. L., et al,(Neuron, 2011, 70, 1071-1084) and such methods are well known in theart.

The PCR amplification products are loaded onto gels, stained withethidium bromide, and subjected to densitometry analysis. Meanintensities from regions of interest (ROI) that correspond to the bandsof interest in the gel are measured.

Gene (or RNA) target quantities obtained by PCR are normalized using theexpression level of a housekeeping gene whose expression is constant,such as GAPDH. Expression of the housekeeping gene (or RNA) is analyzedand measured using the same methods as the target.

Probes and primers are designed to hybridize to a C9ORF72 nucleic acid.Methods for designing RT-PCR probes and primers are well known in theart, and may include the use of software such as PRIMER EXPRESS Software(Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of C9ORF72 nucleic acids can be assessed bymeasuring C9ORF72 protein levels. Protein levels of C9ORF72 can beevaluated or quantitated in a variety of ways well known in the art,such as immunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS). Antibodies directed to a target can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art. Antibodies useful for the detection of mouse,rat, monkey, and human C9ORF72 are commercially available.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of C9ORF72 andproduce phenotypic changes, such as, improved motor function andrespiration. In certain embodiments, motor function is measured byrotarod, grip strength, pole climb, open field performance, balancebeam, hindpaw footprint testing in the animal. In certain embodiments,respiration is measured by whole body plethysmograph, invasiveresistance, and compliance measurements in the animal. Testing may beperformed in normal animals, or in experimental disease models. Foradministration to animals, antisense oligonucleotides are formulated ina pharmaceutically acceptable diluent, such as phosphate-bufferedsaline. Administration includes parenteral routes of administration,such as intraperitoneal, intravenous, and subcutaneous. Calculation ofantisense oligonucleotide dosage and dosing frequency is within theabilities of those skilled in the art, and depends upon factors such asroute of administration and animal body weight. Following a period oftreatment with antisense oligonucleotides, RNA is isolated from CNStissue or CSF and changes in C9ORF72 nucleic acid expression aremeasured.

Targeting C9ORF72

Antisense oligonucleotides described herein may hybridize to a C9ORF72nucleic acid derived from either DNA strand. For example, antisenseoligonucleotides described herein may hybridize to a C9ORF72 antisensetranscript or a C9ORF72 sense transcript. Antisense oligonucleotidesdescribed herein may hybridize to a C9ORF72 nucleic acid in any stage ofRNA processing. Described herein are antisense oligonucleotides that arecomplementary to a pre-mRNA or a mature mRNA. Additionally, antisenseoligonucleotides described herein may hybridize to any element of aC9ORF72 nucleic acid. For example, described herein are antisenseoligonucleotides that are complementary to an exon, an intron, the 5′UTR, the 3′ UTR, a repeat region, a hexanucleotide repeat expansion, asplice junction, an exon:exon splice junction, an exonic splicingsilencer (ESS), an exonic splicing enhancer (ESE), exon 1a, exon 1b,exon 1c, exon 1d, exon 1e, exon 2, exon 3, exon 4, exon 5, exon 6, exon7, exon 8, exon 9, exon 10, exon 11, intron 1, intron 2, intron 3,intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, or intron 10of a C9ORF72 nucleic acid.

In certain embodiments, antisense oligonucleotides described hereinhybridize to all variants of C9ORF72 derived from the sense strand. Incertain embodiments, the antisense oligonucleotides described hereinselectively hybridize to certain variants of C9ORF72 derived from thesense strand. In certain embodiments, the antisense oligonucleotidesdescribed herein selectively hybridize to variants of C9ORF72 derivedfrom the sense strand containing a hexanucleotide repeat expansion. Incertain embodiments, the antisense oligonucleotides described hereinselectively hybridize to pre-mRNA variants containing a hexanucleotiderepeat. In certain embodiments, pre-mRNA variants of C9ORF72 containinga hexanucleotide repeat expansion include SEQ ID NO: 1-3 and 6-10. Incertain embodiments, such hexanucleotide repeat expansion comprises atleast 24 repeats of any of GGGGCC, GGGGGG, GGGGGC, or GGGGCG.

In certain embodiments, the antisense oligonucleotides described hereininhibit expression of all variants of C9ORF72 derived from the sensestrand. In certain embodiments, the antisense oligonucleotides describedherein inhibit expression of all variants of C9ORF72 derived from thesense strand equally. In certain embodiments, the antisenseoligonucleotides described herein preferentially inhibit expression ofone or more variants of C9ORF72 derived from the sense strand. Incertain embodiments, the antisense oligonucleotides described hereinpreferentially inhibit expression of variants of C9ORF72 derived fromthe sense strand containing a hexanucleotide repeat expansion. Incertain embodiments, the antisense oligonucleotides described hereinselectively inhibit expression of pre-mRNA variants containing thehexanucleotide repeat. In certain embodiments, the antisenseoligonucleotides described herein selectively inhibit expression ofC9ORF72 pathogenic associated mRNA variants. In certain embodiments,pre-mRNA variants of C9ORF72 containing a hexanucleotide repeatexpansion include SEQ ID NO: 1-3 and 6-10. In certain embodiments, suchhexanucleotide repeat expansion comprises at least 24 repeats of any ofGGGGCC, GGGGGG, GGGGGC, or GGGGCG. In certain embodiments, thehexanucleotide repeat expansion forms C9ORF72 sense foci. In certainembodiments, antisense oligonucleotides described herein are useful forreducing C9ORF72 sense foci. C9ORF72 sense foci may be reduced in termsof percent of cells with foci as well as number of foci per cell.

C9OFF72 Features

Antisense oligonucleotides described herein may hybridize to any C9ORF72nucleic acid at any state of processing within any element of theC9ORF72 gene. In certain embodiments, antisense oligonucleotidesdescribed herein may target the antisense transcript, e.g., SEQ ID NO:13. In certain embodiments, antisense oligonucleotides described hereinmay hybridize to an exon, an intron, the 5′ UTR, the 3′ UTR, a repeatregion, a hexanucleotide repeat expansion, a splice junction, anexon:exon splice junction, an exonic splicing silencer (ESS), an exonicsplicing enhancer (ESE), exon 1a, exon 1b, exon 1c, exon 1 d, exon 1e,exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10,exon 11, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6,intron 7, intron 8, intron 9, or intron 10. For example, antisenseoligonucleotides may target any of the exons characterized below inTables 1-5 described below. Antisense oligonucleotides described hereinmay also target nucleic acids not characterized below and such nucleicacid may be characterized in GENBANK. Moreover, antisenseoligonucleotides described herein may also target elements other thanexons and such elements as characterized in GENBANK.

TABLE 1 Functional Segments for NM_001256054.1 (SEQ ID NO: 1) Start siteStop site in in reference reference mRNA mRNA to SEQ to SEQ Exon startstop ID ID Number site site NO: 2 NO: 2 exon 1C 1 158 1137 1294 exon 2159 646 7839 8326 exon 3 647 706 9413 9472 exon 4 707 802 12527 12622exon 5 803 867 13354 13418 exon 6 868 940 14704 14776 exon 7 941 105716396 16512 exon 8 1058 1293 18207 18442 exon 9 1294 1351 24296 24353exon 10 1352 1461 26337 26446 exon 11 1462 3339 26581 28458

TABLE 2 Functional Segments for NM_018325.3 (SEQ ID NO: 4) Start siteStop site in in reference reference mRNA mRNA to SEQ to SEQ Exon startstop ID ID Number site site NO: 2 NO: 2 exon 1B 1 63 1510 1572 exon 2 64551 7839 8326 exon 3 552 611 9413 9472 exon 4 612 707 12527 12622 exon 5708 772 13354 13418 exon 6 773 845 14704 14776 exon 7 846 962 1639616512 exon 8 963 1198 18207 18442 exon 9 1199 1256 24296 24353 exon 101257 1366 26337 26446 exon 11 1367 3244 26581 28458

TABLE 3 Functional Segments for NM_145005.5 (SEQ ID NO: 6) Start siteStop site in in reference reference mRNA mRNA to SEQ to SEQ Exon startstop ID ID Number site site NO: 2 NO: 2 exon 1A 1 80 1137 1216 exon 2 81568 7839 8326 exon 3 569 628 9413 9472 exon 4 629 724 12527 12622 exon5B 725 1871 13354 14500 (exon 5 into intron 5)

TABLE 4 Functional Segments for DB079375.1 (SEQ ID NO: 7) Start siteStop site in in reference reference mRNA mRNA to SEQ to SEQ Exon startstop ID ID Number site site NO: 2 NO: 2 exon 1E 1 35 1135 1169 exon 2 36524 7839 8326 exon 3 (EST 525 562 9413 9450 ends before end of fullexon)

TABLE 5 Functional Segments for BU194591.1 (SEQ ID NO: 8) Start siteStop site in in reference reference mRNA mRNA to SEQ to SEQ Exon startstop ID ID Number site site NO: 2 NO: 2 exon 1D 1 36 1241 1279 exon 2 37524 7839 8326 exon 3 525 584 9413 9472 exon 4 585 680 12527 12622 exon5B 681 798 13354 13465 (exon 5 into intron 5)

Certain Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions described herein. In certain embodiments, the individualhas a neurodegenerative disease. In certain embodiments, the individualis at risk for developing a neurodegenerative disease, including, butnot limited to, ALS or FTD. In certain embodiments, the individual hasbeen identified as having a C9ORF72 associated disease. In certainembodiments, the individual has been identified as having a C9ORF72hexanucleotide repeat expansion associated disease. In certainembodiments, provided herein are methods for prophylactically reducingC9ORF72 expression in an individual. Certain embodiments includetreating an individual in need thereof by administering to an individuala therapeutically effective amount of an antisense compound targeted toa C9ORF72 nucleic acid.

In one embodiment, administration of a therapeutically effective amountof an antisense compound targeted to a C9ORF72 nucleic acid isaccompanied by monitoring of C9ORF72 levels in an individual, todetermine an individual's response to administration of the antisensecompound. An individual's response to administration of the antisensecompound may be used by a physician to determine the amount and durationof therapeutic intervention.

In certain embodiments, administration of an antisense compound targetedto a C9ORF72 nucleic acid results in reduction of C9ORF72 expression byat least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 99%, or a range defined by any two of these values. In certainembodiments, administration of an antisense compound targeted to aC9ORF72 nucleic acid results in improved motor function and respirationin an animal. In certain embodiments, administration of a C9ORF72antisense compound improves motor function and respiration by at least15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or99%, or a range defined by any two of these values.

In certain embodiments, administration of an antisense compound targetedto a C9ORF72 antisense transcript results in reduction of C9ORF72antisense transcript expression by at least 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by anytwo of these values. In certain embodiments, administration of anantisense compound targeted to a C9ORF72 antisense transcript results inimproved motor function and respiration in an animal. In certainembodiments, administration of a C9ORF72 antisense compound improvesmotor function and respiration by at least 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by anytwo of these values. In certain embodiments, administration of a C9ORF72antisense compound reduces the number of cells with C9ORF72 antisensefoci and/or the number of C9ORF72 antisense foci per cell.

In certain embodiments, administration of an antisense compound targetedto a C9ORF72 sense transcript results in reduction of a C9ORF72 sensetranscript expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two ofthese values. In certain embodiments, administration of an antisensecompound targeted to a C9ORF72 sense transcript results in improvedmotor function and respiration in an animal. In certain embodiments,administration of a C9ORF72 antisense compound improves motor functionand respiration by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues. In certain embodiments, administration of a C9ORF72 antisensecompound reduces the number of cells with C9ORF72 sense foci and/or thenumber of C9ORF72 sense foci per cell.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to a C9ORF72 nucleic are used for thepreparation of a medicament for treating a patient suffering orsusceptible to a neurodegenerative disease including ALS and FTD.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositionsdescribed herein are co-administered with one or more otherpharmaceutical agents. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease, disorder,or condition as the one or more pharmaceutical compositions describedherein. In certain embodiments, such one or more other pharmaceuticalagents are designed to treat a different disease, disorder, or conditionas the one or more pharmaceutical compositions described herein. Incertain embodiments, such one or more other pharmaceutical agents aredesigned to treat an undesired side effect of one or more pharmaceuticalcompositions described herein. In certain embodiments, one or morepharmaceutical compositions described herein are co-administered withanother pharmaceutical agent to treat an undesired effect of that otherpharmaceutical agent. In certain embodiments, one or more pharmaceuticalcompositions described herein are co-administered with anotherpharmaceutical agent to produce a combinational effect. In certainembodiments, one or more pharmaceutical compositions described hereinare co-administered with another pharmaceutical agent to produce asynergistic effect.

In certain embodiments, one or more pharmaceutical compositionsdescribed herein and one or more other pharmaceutical agents areadministered at the same time. In certain embodiments, one or morepharmaceutical compositions described herein and one or more otherpharmaceutical agents are administered at different times. In certainembodiments, one or more pharmaceutical compositions described hereinand one or more other pharmaceutical agents are prepared together in asingle formulation. In certain embodiments, one or more pharmaceuticalcompositions described herein and one or more other pharmaceuticalagents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition described hereininclude Riluzole (Rilutek), Lioresal (Lioresal), and Dexpramipexole.

In certain embodiments, pharmaceutical agents that may beco-administered with a C9ORF72 antisense transcript specific inhibitordescribed herein include, but are not limited to, an additional C9ORF72inhibitor. In certain embodiments, the co-administered pharmaceuticalagent is administered prior to administration of a pharmaceuticalcomposition described herein. In certain embodiments, theco-administered pharmaceutical agent is administered followingadministration of a pharmaceutical composition described herein. Incertain embodiments the co-administered pharmaceutical agent isadministered at the same time as a pharmaceutical composition describedherein. In certain embodiments the dose of a co-administeredpharmaceutical agent is the same as the dose that would be administeredif the co-administered pharmaceutical agent was administered alone. Incertain embodiments the dose of a co-administered pharmaceutical agentis lower than the dose that would be administered if the co-administeredpharmaceutical agent was administered alone. In certain embodiments thedose of a co-administered pharmaceutical agent is greater than the dosethat would be administered if the co-administered pharmaceutical agentwas administered alone.

In certain embodiments, the co-administration of a second compoundenhances the effect of a first compound, such that co-administration ofthe compounds results in an effect that is greater than the effect ofadministering the first compound alone. In other embodiments, theco-administration results in effects that are additive of the effects ofthe compounds when administered alone. In certain embodiments, theco-administration results in effects that are supra-additive of theeffects of the compounds when administered alone. In certainembodiments, the first compound is an antisense compound. In certainembodiments, the second compound is an antisense compound.

Certain Human Therapeutics

The human C9ORF72 antisense transcript specific antisense compoundsdescribed herein are being evaluated as possible human therapeutics.Various parameters of potency, efficacy, and/or tolerability are beingexamined. Such parameters include in vitro inhibition of C9ORF72antisense transcript; in vitro dose response (IC50); in vivo inhibitionof C9ORF72 antisense transcript in a transgenic animal containing ahuman C9ORF72 transgene in relevant tissues (e.g., brain and/or spinalcord); and/or tolerability in mouse, rat, dog, and/or primate.Tolerability markers that may be measured include blood and serumchemistry parameters, CSF chemistry parameters, body and organ weights,general observations and/or behavioral tests, and/or biochemical markerssuch as GFAP and/or AIF 1. Acute or long term tolerability may bemeasured.

Certain Assays for Measuring C9ORF72 Antisense Transcripts

Certain assays described herein are directed to the reduction of C9ORF72antisense transcript. Additional assays may be used to measure thereduction of C9ORF72 antisense transcript. Additional controls may beused as a baseline for measuring the reduction of C9ORF72 transcript.

Certain Hotspot Regions 1. Nucleobases 196-280 of SEQ ID NO: 13

In certain embodiments, antisense oligonucleotides are designed totarget nucleobases 196-280 of SEQ ID NO: 13. In certain embodiments,nucleobases 196-280 are a hotspot region. In certain embodiments,nucleobases 196-280 are targeted by antisense oligonucleotides. Incertain embodiments, the antisense oligonucleotides are 20 nucleobasesin length. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers.

In certain embodiments, nucleobases 196-280 are targeted by thefollowing ISIS numbers: 687280, 687281, 687282, 687283, 687284, 687285,687286, 687287, 687288, 687289, 687290, 687291, 687292, and 687293.

In certain embodiments, nucleobases 196-280 are targeted by thefollowing SEQ ID NOs: 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, and 82.

In certain embodiments, antisense oligonucleotides targeting nucleobases196-280 achieve at least 45%, at least 46%, at least 47%, at least 48%,at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, or at least 73%reduction of C9ORF72 antisense transcript in vitro.

2. Nucleobases 286-315 of SEQ ID NO: 13

In certain embodiments, antisense oligonucleotides are designed totarget nucleobases 286-315 of SEQ ID NO: 13. In certain embodiments,nucleobases 286-315 are a hotspot region. In certain embodiments,nucleobases 286-315 are targeted by antisense oligonucleotides. Incertain embodiments, the antisense oligonucleotides are 20 nucleobasesin length. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers.

In certain embodiments, nucleobases 286-315 are targeted by thefollowing ISIS numbers: 687277, 687278, and 687279.

In certain embodiments, nucleobases 286-315 are targeted by thefollowing SEQ ID NOs: 66, 67, and 68.

In certain embodiments, antisense oligonucleotides targeting nucleobases286-315 achieve at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, at least 11%, at least 12%, at least 13%, at least 14%, atleast 15%, at least 16%, at least 17%, at least 18%, at least 19%, atleast 20%, at least 21%, at least 22%, at least 23%, at least 24%, atleast 25%, at least 26%, at least 27%, at least 28%, at least 29%, atleast 30%, at least 31%, at least 32%, at least 33%, at least 34%, atleast 35%, at least 36%, at least 37%, at least 38%, at least 39%, atleast 40%, at least 41%, at least 42%, at least 43%, at least 44%, atleast 45%, at least 46%, at least 47%, at least 48%, at least 49%, atleast 50%, at least 51%, at least 52%, at least 53%, at least 54%, atleast 55%, at least 56%, at least 57%, at least 58%, at least 59%, atleast 60%, at least 61%, at least 62%, at least 63%, or at least 64%reduction of C9ORF72 antisense transcript in vitro.

3. Nucleobases 321-415 of SEQ ID NO: 13

In certain embodiments, antisense oligonucleotides are designed totarget nucleobases 321-415 of SEQ ID NO: 13. In certain embodiments,nucleobases 321-415 are a hotspot region. In certain embodiments,nucleobases 321-415 are targeted by antisense oligonucleotides. Incertain embodiments, the antisense oligonucleotides are 20 nucleobasesin length. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers.

In certain embodiments, nucleobases 321-415 are targeted by thefollowing ISIS numbers: 687261, 687262, 687263, 687264, 687265, 687266,687267, 687268, 687269, 687270, 687271, 687272, 687273, 687274, 687275,and 687276.

In certain embodiments, nucleobases 321-415 are targeted by thefollowing SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65.

In certain embodiments, antisense oligonucleotides targeting nucleobases321-415 achieve at least 15%, at least 16%, at least 17%, at least 18%,at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, atleast 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 31%, at least 32%, at least 33%, atleast 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, or at least 70% reduction of C9ORF72 antisense transcript invitro.

4. Nucleobases 451-516 of SEQ ID NO: 13

In certain embodiments, antisense oligonucleotides are designed totarget nucleobases 451-516 of SEQ ID NO: 13. In certain embodiments,nucleobases 451-516 are a hotspot region. In certain embodiments,nucleobases 451-516 are targeted by antisense oligonucleotides. Incertain embodiments, the antisense oligonucleotides are 16, 18, or 20nucleobases in length. In certain embodiments, the antisenseoligonucleotides are gapmers. In certain embodiments, the gapmers are5-10-5 MOE gapmers, 5-8-5 MOE gapmers, 4-8-4 MOE gapmers. In certainembodiments, each nucleoside of the antisense oligonucleotides ismodified with a 2′-MOE substitution. In certain embodiments, theantisense oligonucleotides contain one or more inosine residues.

In certain embodiments, nucleobases 451-516 are targeted by thefollowing ISIS numbers: 687255, 687256, 687257, 687258, 687259, 687260,730389, 730390, 730391, 730392, 730393, 730394, 730395, 730396, 730397,730398, 730399, 730400, 730401, 730402, 730403, 730404, 730405, 730406,730407, 730408, 730409, 730410, 730411, 730412, 737821, 742033, 742034,and 742035.

In certain embodiments, nucleobases 451-516 are targeted by thefollowing SEQ ID NOs: 44, 45, 46, 47, 48, 49, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 98, and 99.

In certain embodiments, antisense oligonucleotides targeting nucleobases451-516 achieve at least 10%, at least 11%, at least 12%, at least 13%,at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, atleast 19%, at least 20%, at least 21%, at least 22%, at least 23%, atleast 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 31%, at least 32%, at least 33%, atleast 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, or at least 78%reduction of C9ORF72 antisense transcript in vitro.

5. Nucleobases 527-588 of SEQ ID NO: 13

In certain embodiments, antisense oligonucleotides are designed totarget nucleobases 527-588 of SEQ ID NO: 13. In certain embodiments,nucleobases 527-588 are a hotspot region. In certain embodiments,nucleobases 527-588 are targeted by antisense oligonucleotides. Incertain embodiments, the antisense oligonucleotides are 20 nucleobasesin length. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers.

In certain embodiments, nucleobases 527-588 are targeted by thefollowing ISIS numbers: 687247, 687248, 687249, 687250, 687251, 687252,687253, and 687254.

In certain embodiments, nucleobases 527-588 are targeted by thefollowing SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, and 43.

In certain embodiments, antisense oligonucleotides targeting nucleobases527-588 achieve at least 21%, at least 22%, at least 23%, at least 24%,at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, atleast 30%, at least 31%, at least 32%, at least 33%, at least 34%, atleast 35%, at least 36%, at least 37%, at least 38%, at least 39%, atleast 40%, at least 41%, at least 42%, at least 43%, at least 44%, atleast 45%, at least 46%, at least 47%, at least 48%, at least 49%, atleast 50%, at least 51%, at least 52%, at least 53%, at least 54%, atleast 55%, at least 56%, at least 57%, at least 58%, at least 59%, atleast 60%, at least 61%, at least 62%, at least 63%, at least 64%, atleast 65%, at least 66%, at least 67%, at least 68%, at least 69%, atleast 70%, at least 71%, at least 72%, or at least 73% reduction ofC9ORF72 antisense transcript in vitro.

6. Nucleobases 608-636 of SEQ ID NO: 13

In certain embodiments, antisense oligonucleotides are designed totarget nucleobases 608-636 of SEQ ID NO: 13. In certain embodiments,nucleobases 608-636 are a hotspot region. In certain embodiments,nucleobases 608-636 are targeted by antisense oligonucleotides. Incertain embodiments, the antisense oligonucleotides are 20 nucleobasesin length. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers.

In certain embodiments, nucleobases 608-636 are targeted by thefollowing ISIS numbers: 687243, 687244, 687245, and 687246.

In certain embodiments, nucleobases 608-636 are targeted by thefollowing SEQ ID NOs: 32, 33, 34, and 35.

In certain embodiments, antisense oligonucleotides targeting nucleobases608-636 achieve at least 20%, at least 21%, at least 22%, at least 23%,at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 31%, at least 32%, at least 33%, atleast 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, or at least 72% reduction ofC9ORF72 antisense transcript in vitro.

7. Nucleobases 704-726 of SEQ ID NO: 13

In certain embodiments, antisense oligonucleotides are designed totarget nucleobases 704-726 of SEQ ID NO: 13. In certain embodiments,nucleobases 704-726 are a hotspot region. In certain embodiments,nucleobases 704-726 are targeted by antisense oligonucleotides. Incertain embodiments, the antisense oligonucleotides are 20 nucleobasesin length. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers.

In certain embodiments, nucleobases 704-726 are targeted by thefollowing ISIS numbers: 687241 and 687242.

In certain embodiments, nucleobases 704-726 are targeted by thefollowing SEQ ID NOs: 30 and 31.

In certain embodiments, antisense oligonucleotides targeting nucleobases704-726 achieve at least 19%, at least 20%, or at least 21% reduction ofC9ORF72 antisense transcript in vitro.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions, and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1: Antisense Inhibition of C9ORF72 Antisense Transcript withOligonucleotides

Antisense oligonucleotides (ASOs) targeting the human C9ORF72 antisensetranscript were made and tested for inhibition of target transcriptexpression in vitro. All of the ASOs in the table below except IsisNumbers 742033 and 742035 are 5-10-5 MOE gapmers with phosphorothioateinternucleoside linkages throughout the gapmers. They are 20 nucleosidesin length, with a central gap segment consisting of ten2′-deoxynucleosides that is flanked by wing segments in the 5′ directionand the 3′ direction consisting of five nucleosides each. Eachnucleoside in the 5′ and 3′ wing segments has a 2′-MOE modification. Allcytosine residues throughout each gapmer are 5 methylcytosines. Isis No.742033 is a 5-10-5 MOE gapmer with phosphodiester (“o”) andphosphorothioate (“s”) internucleoside linkages arranged in order from5′ to 3′: soooossssssssssooss. Isis No. 742035 is a 5-8-5 MOE gapmerwith a central gap segment consisting of eight 2′-deoxynucleosides andinternucleoside linkages arranged in order from 5′ to 3′:sooosssssssssooss. All cytosine residues in Isis Numbers 742033 and742035 are 5-methylcytosines. Isis Numbers 742033 and 742035 alsocontain inosine residues, indicated by “I”. In the table below, “Start”indicates the 5′-most nucleoside to which the gapmer is targeted in thetarget transcript sequence. “Stop” indicates the 3′-most nucleoside towhich the gapmer is targeted in the target transcript sequence. Eachgapmer listed in the table below is targeted to a putative antisensetranscript sequence, designated herein as SEQ ID NO: 13. The sequence ofSEQ ID NO: 13 is complementary to nucleotides 1159 to 1929 of SEQ ID NO:2 (the complement of GENBANK Accession No. NT 008413.18 truncated fromnucleotides 27535000 to 27565000) except that SEQ ID NO: 13 has two morehexanucleotide repeats than SEQ ID NO: 2. The sequence of thehexanucleotide repeat is GGCCCC in SEQ ID NO: 13 and GGGGCC in SEQ IDNO: 2. Thus, SEQ ID NO: 13 is 12 nucleotides longer than nucleotides1159 to 1929 of SEQ ID NO: 2, to which it is complementary. Isis No.129700 is a negative control ASO that does not target the C9ORF72antisense transcript.

bEND cells were cultured in 24 well plates at 45,000-50,000 cells/well24 hours before the first of two transfections. The cells were firsttransfected with 0.2 μg/well of a plasmid expressing the C9ORF72antisense transcript (SEQ ID NO: 13) and 0.5 μL Lipofectamine 2000 (LifeTechnologies, Carlsbad, Calif.) in OptiMEM medium. Four to six hourslater, the media was replaced. 24 hours after the first transfection(18-20 hours after media replacement), the bEND cells were transfectedwith 25 nM of an ASO listed in the table below and 0.5 μL Lipofectamine2000 in OptiMEM medium or with no ASO. Just prior to transfection, ASOscomprising a GGGGCC repeat with or without guanosine to inosinesubstitutions (Isis Numbers 687258, 687259, 687260, 742033, and 742035)were heated at 90° C. for five minutes, then placed on ice brieflybefore dilution in OptiMEM. Total RNA was isolated from the cells 24hours after the second transfection using TRIzol (Life Technologies)according to the manufacturer's directions. Two DNase reactions wereperformed, one on the column during RNA purification, and one afterpurification using TURBO DNase (Life Technologies).

Strand specific RT-qPCR was performed on the isolated RNA to generateand amplify C9ORF72 antisense cDNA using one or two of three differentprimer sets, LTS01222, LTS01221, and C9ATS3′-1. The LTS01222 sequencesare: RT primer: CGACTGGAGCACGAGGACACTGAAAAGATGACGCTTGGTGTGTCA (SEQ IDNO: 14), forward PCR primer: CCCACACCTGCTCTTGCTAGA (SEQ ID NO: 15),reverse PCR primer: CGACTGGAGCACGAGGACACTG (SEQ ID NO: 16), and probe:CCCAAAAGAGAAGCAACCGGGCA (SEQ ID NO: 17). The LTS01221 sequences are: RTprimer: CGACTGGAGCACGAGGACACTGACGGCTGCCGGGAAGA (SEQ ID NO: 18), forwardPCR primer: AGAAATGAGAGGGAAAGTAAAAATGC (SEQ ID NO: 19), reverse PCRprimer: CGACTGGAGCACGAGGACACTG (SEQ ID NO: 20), and probe:AGGAGAGCCCCCGCTTCTACCCG (SEQ ID NO: 21). The C9ATS3′-1 sequences are: RTprimer: CGACTGGAGCACGAGGACACTGACGCTGAGGGTGAACAAGAA (SEQ ID NO: 22),forward PCR primer: GAGTTCCAGAGCTTGCTACAG (SEQ ID NO: 23), reverse PCRprimer: CGACTGGAGCACGAGGACACTG (SEQ ID NO: 24), and probe:CTGCGGTTGTTTCCCTCCTTGTTT (SEQ ID NO: 25). RT-qPCR was also performed onthe isolated RNA using Express One-Step Superscript qRT-PCR Kit (LifeTechnologies, Carlsbad, Calif.) according to manufacturer's instructionsto generate and amplify GAPDH cDNA, as a control, using forward PCRprimer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 26), reverse PCR primer:GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 27), and probe:AAGGCCGAGAATGGGAAGCTTGTCATC (SEQ ID NO: 28). The resulting C9ORF72antisense levels were normalized to GAPDH. These normalized values forC9ORF72 antisense transcript expression in cells treated with an ASOwere then compared to the normalized values for C9ORF72 antisensetranscript expression in control cells that were transfected with theC9ORF72 antisense plasmid but not an ASO. The results for each primerprobe set are shown in the table below as percent inhibition of C9ORF72antisense transcript expression relative to the control cells that werenot transfected with an ASO. A result of 0% inhibition indicates thatthe C9ORF72 antisense transcript levels were equal to that of controlcells that were not transfected with an ASO. A negative value for %inhibition indicates that the C9ORF72 antisense transcript levels werehigher than that of control cells that were not transfected with an ASO.A result of “n/a” indicates that the corresponding primer probe set wasnot used to analyze the indicated sample. The results show that manyASOs inhibited human C9ORF72 antisense transcript expression. Theabsolute inhibition results varied across different primer probe sets,but the relative potencies of the ASOs were very similar acrossdifferent primer probe sets.

TABLE 6 C9ORF72 Antisense RNA Inhibition by Antisense OligonucleotidesSEQ Isis LTS LTS C9AT ID No. Start Stop Sequence 01222 01221 S3′-1 No.129700 n/a n/a TAGTGCGGACCTACCCACGA 98 92 101 29 687241 707 726GGTGTGTCAGCCGTCCCTGC n/a 19 21 30 687242 704 723 GTGTCAGCCGTCCCTGCTGCn/a −44 −1 31 687243 617 636 TGTTTTTCCCACCCTCTCTC 70 20 n/a 32 687244614 633 TTTTCCCACCCTCTCTCCCC 68 21 n/a 33 687245 611 630TCCCACCCTCTCTCCCCACT 72 −5 n/a 34 687246 608 627 CACCCTCTCTCCCCACTACT 5917 n/a 35 687247 569 588 GAGGGTGAACAAGAAAAGAC 71 51 n/a 36 687248 557576 GAAAAGACCTGATAAAGATT 50 30 n/a 37 687249 542 561AGATTAACCAGAAGAAAACA 54 21 n/a 38 687250 539 558 TTAACCAGAAGAAAACAAGG 6335 n/a 39 687251 536 555 ACCAGAAGAAAACAAGGAGG 73 41 n/a 40 687252 533552 AGAAGAAAACAAGGAGGGAA 44 33 n/a 41 687253 530 549AGAAAACAAGGAGGGAAACA 68 47 n/a 42 687254 527 546 AAACAAGGAGGGAAACAACC 5733 n/a 43 687255 497 516 GCAAGCTCTGGAACTCAGGA 51 n/a n/a 44 687256 494513 AGCTCTGGAACTCAGGAGTC 57 n/a n/a 45 687257 483 502TCAGGAGTCGCGCGCTAGGG 68 n/a n/a 46 687258 480 499 GGAGTCGCGCGCTAGGGGCC54 n/a n/a 47 687259 477 496 GTCGCGCGCTAGGGGCCGGG 63 n/a n/a 48 687260474 493 GCGCGCTAGGGGCCGGGGCC 44 n/a n/a 49 687261 396 415GGGCTGCGGTTGCGGTGCCT 58 n/a 56 50 687262 391 410 GCGGTTGCGGTGCCTGCGCC 55n/a 55 51 687263 386 405 TGCGGTGCCTGCGCCCGCGG 51 n/a 53 52 687264 381400 TGCCTGCGCCCGCGGCGGCG 40 n/a 35 53 687265 376 395GCGCCCGCGGCGGCGGAGGC 16 n/a 15 54 687266 371 390 CGCGGCGGCGGAGGCGCAGG 45n/a 40 55 687267 366 385 CGGCGGAGGCGCAGGCGGTG 46 n/a 45 56 687268 361380 GAGGCGCAGGCGGTGGCGAG 29 n/a 39 57 687269 356 375GCAGGCGGTGGCGAGTGGGT 51 n/a 52 58 687270 351 370 CGGTGGCGAGTGGGTGAGTG 56n/a 55 59 687271 346 365 GCGAGTGGGTGAGTGAGGAG 70 n/a 69 60 687272 341360 TGGGTGAGTGAGGAGGCGGC 69 n/a 69 61 687273 336 355GAGTGAGGAGGCGGCATCCT 56 n/a 51 62 687274 331 350 AGGAGGCGGCATCCTGGCGG 49n/a 46 63 687275 326 345 GCGGCATCCTGGCGGGTGGC 54 n/a 51 64 687276 321340 ATCCTGGCGGGTGGCTGTTT 66 n/a 64 65 687277 296 315TCGGCTGCCGGGAAGAGGCG 62 n/a 64 66 687278 291 310 TGCCGGGAAGAGGCGCGGGT 46n/a 41 67 687279 286 305 GGAAGAGGCGCGGGTAGAAG 6 n/a 8 68 687280 261 280GCTCTCCTCAGAGCTCGACG 48 n/a 50 69 687281 256 275 CCTCAGAGCTCGACGCATTT 48n/a 48 70 687282 251 270 GAGCTCGACGCATTTTTACT 57 n/a 55 71 687283 246265 CGACGCATTTTTACTTTCCC 66 n/a 62 72 687284 241 260CATTTTTACTTTCCCTCTCA 66 n/a 67 73 687285 236 255 TTACTTTCCCTCTCATTTCT 64n/a 61 74 687286 231 250 TTCCCTCTCATTTCTCTGAC 60 n/a 56 75 687287 226245 TCTCATTTCTCTGACCGAAG 64 n/a 45 76 687288 221 240TTTCTCTGACCGAAGCTGGG 70 n/a 67 77 687289 216 235 CTGACCGAAGCTGGGTGTCG 71n/a 66 78 687290 211 230 CGAAGCTGGGTGTCGGGCTT 64 n/a 65 79 687291 206225 CTGGGTGTCGGGCTTTCGCC 73 n/a 68 80 687292 201 220TGTCGGGCTTTCGCCTCTAG 66 n/a 67 81 687293 196 215 GGCTTTCGCCTCTAGCGACT 70n/a 71 82 742033 456 475 CCGGGICCGIGGCCIGGGCC 74 36 50 83 462 481 742035454 471 GGCCGIGGCCGGIGCCGG 37 n/a 18 84 460 477 466 483

Example 2: Dose Dependent Inhibition of C9ORF72 Antisense Transcriptwith an Oligonucleotide Targeting a Hexanucleotide Repeat

Isis No. 742033 (see Example 1) was tested for dose dependent inhibitionof C9ORF72 antisense transcript expression in vitro. bEND cells werecultured and treated as described in Example 1. During the secondtransfection, cells received Isis No. 742033 at a concentration listedin the table below or they received no ASO as a control. Total RNA wasisolated and analyzed as described in Example 1 using primer probe setC9ATS3′-1.

TABLE 7 C9ORF72 Antisense RNA Inhibition by Isis No. 742033Concentration of Isis No. 742033 (nM) % Inhibition 3.125 39 6.25 65 12.568 25.0 72

Example 3: Antisense Inhibition of C9ORF72 Antisense Transcript withOligonucleotides

Antisense oligonucleotides (ASOs) targeting the human C9ORF72 antisensetranscript were made and tested for inhibition of C9ORF72 antisensetranscript expression in vitro. ASOs 730401-730406 in the table beloware 5-8-5 MOE gapmers with phosphorothioate internucleoside linkagesthroughout the gapmers. ASOs 730407-730412 in the table below are 4-8-4MOE gapmers with phosphorothioate internucleoside linkages throughoutthe gapmers. ASOs 730401-730412 all have a central gap segmentconsisting of eight 2′-deoxynucleosides that is flanked by wing segmentsin the 5′ direction and the 3′ direction consisting of four or fivenucleosides each. Each nucleoside in the 5′ and 3′ wing segments has a2′-MOE modification. All cytosine residues throughout each gapmer are5-methylcytosines. In the table below, “Start” indicates the 5′-mostnucleoside to which the gapmer is targeted in the target transcriptsequence. “Stop” indicates the 3′-most nucleoside to which the gapmer istargeted in the target transcript sequence. Each gapmer listed in thetable below is targeted to a putative antisense transcript sequence,designated herein as SEQ ID NO: 13. The sequence of SEQ ID NO: 13 iscomplementary to nucleotides 1159 to 1929 of SEQ ID NO: 2 (thecomplement of GENBANK Accession No. NT 008413.18 truncated fromnucleotides 27535000 to 27565000) except that SEQ ID NO: 13 has two morehexanucleotide repeats than SEQ ID NO: 2. The sequence of thehexanucleotide repeat is GGCCCC in SEQ ID NO: 13 and GGGGCC in SEQ IDNO: 2. Thus, SEQ ID NO: 13 is 12 nucleotides longer than nucleotides1159 to 1929 of SEQ ID NO: 2, to which it is complementary. Isis No.129700 is a negative control ASO that does not target the C9ORF72antisense transcript, and 742035 was included for comparison, as it wasdescribed and tested in Example 1.

bEND cells were cultured and treated as described in Example 1. Strandspecific RT-qPCR was performed on the isolated RNA, as described inExample 1, using the primer sets LTS01222 and LTS01221. The resultingnormalized C9ORF72 antisense levels were then compared to the normalizedvalues for C9ORF72 antisense transcript expression in control cells thatwere transfected with the C9ORF72 antisense plasmid but not an ASO. Theresults for each primer probe set are shown in the table below aspercent inhibition of C9ORF72 antisense transcript expression relativeto the control cells that were not transfected with an ASO. The valuesin the table below are the averages of two separate experiments. Aresult of 0% inhibition indicates that the C9ORF72 antisense transcriptlevels were equal to that of control cells that were not transfectedwith an ASO. A negative value for % inhibition indicates that theC9ORF72 antisense transcript levels were higher than that of controlcells that were not transfected with an ASO. A result of “n/a” indicatesthat the corresponding primer probe set was not used to analyze theindicated sample. The results show that many ASOs inhibited humanC9ORF72 antisense transcript expression. The absolute inhibition resultsvaried across different primer probe sets, but the relative potencies ofthe ASOs were similar across different primer probe sets.

TABLE 8 Inhibition of C9ORF72 Antisense Transcript byAntisense Oligonucleotides SEQ Isis LTS LTS ID No. Start Stop Sequence01222 01221 No. 129700 n/a n/a TAGTGCGGACCTACCCACGA 11 11 29 730401 456473 GGGGCCGGGGCCGGGGCC 71 38 85 462 479 468 485 730402 451 468CGGGGCCGGGGCCGGGGC 67 10 86 457 474 463 480 730403 452 469CCGGGGCCGGGGCCGGGG 64 21 87 458 475 464 481 730404 459 476GCCGGGGCCGGGGCCGGG 65 34 88 465 482 730405 454 471 GGCCGGGGCCGGGGCCGG 4922 89 460 477 466 483 730406 455 472 GGGCCGGGGCCGGGGCCG 41 12 90 461 478467 484 730407 456 471 GGCCGGGGCCGGGGCC 78 44 91 462 477 468 483 730408451 466 GGGCCGGGGCCGGGGC 68 24 92 457 472 463 478 469 484 730409 452 467GGGGCCGGGGCCGGGG 66 38 93 458 473 464 479 470 485 730410 453 468CGGGGCCGGGGCCGGG 67 38 94 459 474 465 480 730411 454 469CCGGGGCCGGGGCCGG 63 16 95 460 475 466 481 730412 455 470GCCGGGGCCGGGGCCG 73 38 96 461 476 467 482 742035 454 471GGCCGIGGCCGGIGCCGG 68 32 84 460 477 466 483

Example 4: Antisense Inhibition of C9ORF72 Antisense Transcript withOligonucleotides

Antisense oligonucleotides (ASOs) described in Example 1 were tested forinhibition of C9ORF72 antisense transcript expression in vitro using acell line in which a CMV promoter was installed to drive the expressionof the endogenous C9ORF72 antisense gene via CRISPR/Cas9 technology.

The targeting portion of a single guide RNA (sgRNA) of the sequence5′-GACAAGGGTACGTAATCTGTC-3′, designated herein as SEQ ID NO: 97, wasdesigned to target a site 1,020 base pairs downstream of the C9ORF72hexanucleotide repeat. An NGG PAM motif is present at the 3′ end of thetarget site. The targeting portion of the sgRNA was inserted into adual-expression plasmid to generate the full-length sgRNA of thesequence: 5′-GACAAGGGTACGTAATCTGTCTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT-3′, designated herein as SEQID NO.: 101 and Cas9 nuclease.

The donor plasmid, containing a 922 base pair 5′ homology arm, reverseCMV (CMVr), and 988 base pair 3′ homology arm, was generated in pCR4(Life Technologies) backbone plasmid by Gibson Assembly. The homologyarm sequences were designed to center around the CRISPR/Cas9 cleavagesite and were constructed using PCR primers:5′-TAGTCCTGCAGGTTTAAACGAATTCGTGAGTGAGGAGGCGGCA-3′, forward primer, SEQID NO: 102, and 5′-AGCAGAGCTCAGATTACGTACCCTTGTTGTGAACAAC-3′, reverseprimer, SEQ ID NO: 103, for the 5′ arm; and5′-CAATGTCAACGTCTGGCATTACTTCTACTTTTG-3′, forward primer, SEQ ID NO: 104,and 5′-TAGGGCGAATTGAATTTAGCGGCCGCACTGGCAGGATCATAGC-3′, reverse primer,SEQ ID NO: 105, for the 3′ arm. The CMVr sequence was amplified frompCDNA3.1 using PCR primers: 5′-TACGTAATCTGAGCTCTGCTTATATAGACC-3′,forward primer, SEQ ID NO: 106, and5′-AATGCCAGACGTTGACATTGATTATTGACTAGTTATTAATAG-3′, reverse primer, SEQ IDNO: 107.

Neuroblast SH-SY5Y cells (Sigma-Aldrich) were cultured in a 1:1 mixtureof MEM:F-12 (Life Technologies) supplemented with 10% FBS, 25 mM HEPESand Antibiotic-Antimycotic. C9orf72 CRISPR/Cas9 activity was assessed bymeasuring indel frequency using SURVEYOR mutation detection assay(Integrated DNA Technologies) with forward primer:5′-GTTAGGCTCTGGGAGAGTAGTTG-3′, SEQ ID NO: 108, and reverse primer:5′-CCTGGAGCAGGTAAATGCTGG-3′, SEQ ID NO: 109. To generate SH-SY5Y cellsexpressing C9orf72 antisense transcript, SH-SY5Y cells were transfectedwith a plasmid expressing C9ORF72 CRISPR sgRNA and Cas9 and with a CMVrdonor plasmid. Furthermore, the cells were co-transfected with a plasmidexpressing EGFP, then single-cell sorted by FACS into 96-well plates.RT-qPCR was performed to screen for increased C9ORF72 antisense RNA.Positive clones were isolated and validated by PCR using primers insideCMVr and outside the 5′ and 3′ arms, respectively. Amplicons werefurther validated by sequencing to confirm on-target insertion of CMVr.Confirmation of single or double allele targeting was obtained by PCRwith primers used in the SURVEYOR assay. Sequencing showed that theC9ORF72 antisense transcript contains 2 full hexanucleotide repeats.

The engineered SH-SY5Y cells were plated at 30,000 cells per well andelectroporated at 140V with 10 μM ASO. 24 hours later cells were lysed.Strand specific RT-qPCR was performed on the isolated RNA, as describedin Example 1, using the primer probe sets LTS01221 or C9ATS3′-1. Theresulting normalized C9ORF72 antisense levels were then compared to thenormalized values for C9ORF72 antisense transcript expression in controlcells that were transfected with neither the C9ORF72 antisense plasmidnor an ASO. The results for each primer probe set are shown in the tablebelow as percent inhibition of C9ORF72 antisense transcript expressionrelative to the control cells. A result of 0% inhibition indicates thatthe C9ORF72 antisense transcript levels were equal to that of controlcells that were not transfected with an ASO. A negative value for %inhibition indicates that the C9ORF72 antisense transcript levels werehigher than that of control cells that were not transfected with an ASO.A result of “n/a” indicates that the corresponding primer probe set wasnot used to analyze the indicated sample. The results show that manyASOs inhibited human C9ORF72 antisense transcript expression.

TABLE 9 Inhibition of C90RF72 Antisense Transcript by AntisenseOligonucleotides Isis No. LTS 01221 C9ATS3′-1 SEQ ID No. 129700 22 −4 29687241 68 n/a 30 687242 60 n/a 31 687243 54 n/a 32 687244 47 n/a 33687245 53 n/a 34 687246 52 n/a 35 687247 53 n/a 36 687248 14 n/a 37687249 6 n/a 38 687250 12 n/a 39 687251 53 n/a 40 687252 32 n/a 41687253 21 n/a 42 687254 20 n/a 43 687255 81 n/a 44 687256 78 n/a 45687257 63 n/a 46 687258 54 n/a 47 687259 52 n/a 48 687260 23 n/a 49687261 9 n/a 50 687262 41 n/a 51 687263 24 n/a 52 687264 42 n/a 53687265 25 n/a 54 687266 51 n/a 55 687267 20 n/a 56 687268 24 n/a 57687269 20 n/a 58 687270 36 n/a 59 687271 10 n/a 60 687272 47 n/a 61687273 50 n/a 62 687274 66 n/a 63 687275 42 n/a 64 687276 48 n/a 65687277 n/a 40 66 687278 n/a 38 67 687279 n/a −24 68 687280 n/a 60 69687281 n/a 69 70 687282 n/a 32 71 687283 n/a 69 72 687284 n/a 47 73687285 n/a 28 74 687286 n/a 67 75 687287 n/a 71 76 687288 n/a 65 77687289 n/a 63 78 687290 62 48 79 687291 75 71 80 687292 77 68 81 68729376 72 82 742033 6 26 83

Example 5: Dose Dependent Inhibition of C9ORF72 Antisense Transcriptwith Oligonucleotides

Isis Numbers 687241, 687255, 687256, 687280, 687281, 687283, 687286,687287, 687288, 687289, 687291, 687292, and 687293 (see Example 1) weretested for dose dependent inhibition of C9ORF72 antisense transcriptexpression in vitro. The SH-SY5Y cells described in Example 4 werecultured at 30,000 cells per well and electroporated at 140 V treatedwith an oligonucleotide at a concentration listed in the tables below orthey received no ASO as a control. 24 hours after electroporation, cellswere lysed and total RNA was isolated and analyzed by RT-qPCR usingprimer probe set LTS01221 (Tables 10 and 11) or C9ATS3′-1 (Table 12).The results are shown below for each antisense oligonucleotideconcentration, and half maximal inhibitory concentrations werecalculated using Prism software (Graphpad).

TABLE 10 Dose Dependent C9ORF72 Antisense Transcript InhibitionConcentration % IC₅₀ Isis No. (μM) Inhibition (μM) 687241 0.625 −5 3.491.25 17 2.5 34 5 52 10 66 20 54 687291 0.625 20 3.17 1.25 15 2.5 31 5 4710 67 20 71

TABLE 11 Dose Dependent C9ORF72 Antisense Transcript Inhibition Dose %IC₅₀ Isis No. (nM) Inhibition (μM) 687255 0.625 9 2.34 1.25 20 2.5 39 557 10 62 20 69 687256 0.625 15 2.78 1.25 19 2.5 37 5 46 10 56 20 68687291 0.625 24 1.72 1.25 24 2.5 41 5 63 10 62 20 70

TABLE 12 Dose Dependent C9ORF72 Antisense Transcript Inhibition Dose %IC₅₀ Isis No. (nM) Inhibition (μM) 687280 0.625 19 2.87 1.25 18 2.5 39 561 10 69 20 66 687281 0.625 13 5.21 1.25 12 2.5 26 5 39 10 60 20 69687283 0.625 16 4.98 1.25 31 2.5 23 5 38 10 53 20 70 687286 0.625 −54.31 1.25 8 2.5 29 5 51 10 65 20 71 687287 0.625 5 3.10 1.25 24 2.5 25 564 10 73 20 85 687288 0.625 10 3.52 1.25 12 2.5 31 5 58 10 66 20 78687289 0.625 14 5.43 1.25 14 2.5 23 5 43 10 61 20 60 687291 0.625 232.09 1.25 33 2.5 35 5 67 10 75 20 77 687292 0.625 19 3.94 1.25 11 2.5 255 56 10 68 20 65 687293 0.625 23 2.15 1.25 25 2.5 44 5 63 10 77 20 75

Example 6: Effect of Antisense Oligonucleotides Targeting C9ORF72Antisense Transcript on RNA Foci

Antisense oligonucleotides described above and those described in thetables below can be tested for their effects on C9ORF72 antisense fociin C9ORF72 ALS/FTD patient fibroblast lines. The antisenseoligonucleotides listed in Tables 13 and 14 below target thehexanucleotide repeat of the C9ORF72 antisense transcript. Eachnucleoside of the antisense oligonucleotides in Table 13 below ismodified with a 2′-MOE substitution. All of the cytosines are5-methylcytosines, and all of the internucleoside linkages arephosphorothioate linkages. The motifs and internucleoside linkages ofthe oligonucleotides in Table 14 are shown in the table. Thesubstitution or lack thereof at the 2′-position of each nucleoside isdenoted as “d”, meaning 2′-deoxy, or “e”, meaning 2′-MOE. Eachinternucleoside linkage is denoted as “o”, meaning phosphodiester, or“s”, meaning phosphorothioate. All of the cytosines in Table 14 are5-methylcytosines. The oligonucleotides in Table 14 also contain inosineresidues, indicated by “I”.

TABLE 13 Fully modified antisense oligonucleotidestargeting the antisense transcript of C9ORF72 SEQ ID Isis No. SequenceNo. 730389 GGGGCCGGGGCCGGGGCC 85 730390 CGGGGCCGGGGCCGGGGC 86 730391CCGGGGCCGGGGCCGGGG 87 730392 GCCGGGGCCGGGGCCGGG 88 730393GGCCGGGGCCGGGGCCGG 89 730394 GGGCCGGGGCCGGGGCCG 90 730395GGCCGGGGCCGGGGCC 91 730396 GGGCCGGGGCCGGGGC 92 730397 GGGGCCGGGGCCGGGG93 730398 CGGGGCCGGGGCCGGG 94 730399 CCGGGGCCGGGGCCGG 95 730400GCCGGGGCCGGGGCCG 96

TABLE 14Antisense oligonucleotides targeting the antisense transcript of C9ORF72SEQ Isis Internucleoside ID No. Sequence (5′ to 3′) Motif (5′ to 3′)linkages (5′ to 3′) No. 737821 CCGIGGCCGIGGCCGIGGCC eeedeeeeedeeeeedeeeessososssososssososs 98 742034 GGCCGIGGCCGIGGCCGG eeeeedeeeeedeeeeeessssososssosossss 99

C9ORF72 antisense foci are visualized using fluorescent in situhybridization with a fluorescently labeled Locked Nucleic Acid (LNA)probe targeting the hexanucleotide repeat containing C9ORF72 antisensetranscript (Exiqon, Inc. Woburn Mass.). The sequence of the probe ispresented in the table below. The probe was labeled with fluorescent 5′TYE-563. A 5′ TYE-563-labeled fluorescent probe targeting CUG repeats isused as a negative control.

TABLE 15 LNA probes to the C9ORF72 antisense transcriptcontaining the hexanucleotide repeat SEQ Description ID Target of probeSequence NO GGCCCC Repeat Fluorescent TYE563- 93 of the LNA ProbeGGGGCCGGGGCCGGGG Antisense Transcript CUG Repeat Fluorescent TYE563- 100LNA Probe CAGCAGCAGCAGCAGCAGC

All hybridization steps were performed under RNase-free conditions.Patient fibroblast cells were plated into chamber slides. 24 hourslater, they were washed in PBS and transfected with 25 nM of an Isisantisense oligonucleotide in the table below or a negative control ASOthat does not target any C9ORF72 RNA using 1 μl/ml Cytofectintransfection reagent (Genlantis, San Diego, Cat #T610001). Cells wereincubated for 4 hours at 37° C. and 5% CO₂, before the medium wasreplaced with Dulbecco's modified Eagle medium (DMEM) supplemented with20% tetracycline-free FBS and 2% penicillin/streptomycin and 1%amphotericin B. 24 hours after transfection, the cells were fixed in 4%PFA, then immediately permeabilized in 0.2% Triton X-100 (Sigma Aldrich#T-8787) in PBS for 10 minutes, washed twice in PBS for 5 minutes,dehydrated with ethanol, and air dried. The slides were heated in 400 μLhybridization buffer (50% deionized formamide, 2×SCC, 50 mM SodiumPhosphate, pH 7, and 10% dextran sulphate) at 66° C. for 20-60 minutesunder floating RNase-free coverslips in a chamber humidified withhybridization buffer. Probes were denatured at 80° C. for 75 seconds andreturned immediately to ice before diluting with hybridization buffer(40 nM final concentration). The incubating buffer was replaced with theprobe-containing mix (400 μL per slide), and slides were hybridizedunder floating coverslips for 12-16 hours in a sealed, light-protectedchamber.

After hybridization, floating coverslips were removed and slides werewashed at room temperature in 0.1% Tween-20/2X SCC for 5 minutes beforebeing subjected to three 10-minute stringency washes in 0.1×SCC at 65°C. The slides were then dehydrated through ethanol and air dried.

Primary visualization for quantification and imaging of foci wasperformed at 100× magnification using a Nikon Eclipse Ti confocalmicroscope system equipped with a Nikon CFI Apo TIRF 100X Oil objective(NA 1.49). Most foci are intra-nuclear but are also occasionally foundin the cytoplasm. Treatment with RNase A, but not DNase I, eliminatedthe C9ORF72 antisense foci, demonstrating that they are comprisedprimarily of RNA. The foci in the fibroblasts were counted, and the datais presented in the table below as the number of foci per positive celland the number of foci per cell overall. (A positive cell is a cell thathas at least one focus.) The data in the table below show that treatmentwith the antisense oligonucleotides targeting the antisense C9ORF72transcript, listed in the table below, decreased both the number ofcells with at least one focus (foci per cell) and the number of fociwithin cells that still had at least one focus (foci per positive cell).

TABLE 16 Antisense C9ORF72 foci in patient fibroblasts Foci Foci IsisNo. per positive cell per cell Negative 2.99 1.50 control ASO 7378212.73 1.00 742033 1.68 0.46 742034 1.38 0.22 742035 1.69 0.53

1. A compound comprising a C9ORF72 antisense transcript specificinhibitor, wherein the C9ORF72 antisense transcript specific inhibitoris a modified oligonucleotide consisting of 12-30 linked nucleosides,wherein at least one nucleoside of the antisense oligonucleotidecomprises a hypoxanthine nucleobase, wherein the modifiedoligonucleotide has a nucleobase sequence that is at least 90%complementary to a C9ORF72 antisense transcript, and wherein themodified oligonucleotide comprises at least one modified internucleosidelinkage and/or at least one modified nucleoside comprising a modifiedsugar.
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. The compound ofclaim 1, wherein the modified oligonucleotide consists of 16-25 linkednucleosides.
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled) 10.The compound of claim 1, wherein the modified oligonucleotide has anucleobase sequence that is 100% complementary to a C9ORF72 antisensetranscript.
 11. The compound of claim 1, wherein the C9ORF72 antisensetranscript has the nucleobase sequence of SEQ ID NO:
 13. 12. Thecompound of claim 1, wherein the modified oligonucleotide has anucleobase sequence comprising at least 16 contiguous nucleobases of asequence selected from SEQ ID NO: 30-96, 98, or
 99. 13. (canceled) 14.The compound of claim 1, wherein the modified oligonucleotide comprisesat least one modified internucleoside linkage.
 15. The compound of claim14, wherein at least one modified internucleoside linkage is aphosphorothioate internucleoside linkage.
 16. The compound of claim 14,wherein each modified internucleoside linkage is a phosphorothioateinternucleoside linkage.
 17. The compound of claim 14 wherein themodified oligonucleotide comprises at least one phosphodiesterinternucleoside linkage.
 18. (canceled)
 19. (canceled)
 20. (canceled)21. The compound of claim 1, wherein at least one nucleoside of themodified oligonucleotide comprises a modified sugar.
 22. (canceled) 23.(canceled)
 24. The compound of claim 21, wherein at least one modifiedsugar comprises a 2′-O-methoxyethyl group.
 25. The compound of claim 1,wherein the modified oligonucleotide is a gapmer. 26-92. (canceled) 93.The compound of claim 1, wherein the modified oligonucleotide is asingle-stranded modified oligonucleotide. 94-145. (canceled)
 146. Acompound comprising a C9ORF72 antisense transcript specific inhibitor,wherein the C9ORF72 antisense transcript specific inhibitor is amodified oligonucleotide consisting of 12-30 linked nucleosides, whereinthe modified oligonucleotide has a nucleobase sequence comprising atleast 12 contiguous nucleobases that are complementary to ahexanucleotide repeat expansion in a C9ORF72 antisense transcript, andwherein at least one nucleoside of the modified oligonucleotidecomprises a modified sugar.
 147. The compound of claim 146, wherein themodified oligonucleotide consists of 16-25 linked nucleosides.
 148. Thecompound of claim 146, wherein the modified oligonucleotide has anucleobase sequence that is at least 90% complementary to the C9ORF72antisense transcript.
 149. The compound of claim 146, wherein themodified oligonucleotide has a nucleobase sequence that is 100%complementary to the C9ORF72 antisense transcript.
 150. The compound ofclaim 146, wherein the modified oligonucleotide has a nucleobasesequence comprising at least 16 contiguous nucleobases of a sequenceselected from SEQ ID NOs: 47-49, 85-96, 98, or
 99. 151. The compound ofclaim 146, wherein the modified oligonucleotide comprises at least onemodified internucleoside linkage.
 152. The compound of claim 151,wherein at least one modified internucleoside linkage is aphosphorothioate internucleoside linkage.
 153. The compound of claim151, wherein each modified internucleoside linkage is a phosphorothioateinternucleoside linkage.
 154. The compound of claim 151, wherein themodified oligonucleotide comprises at least one phosphodiesterinternucleoside linkage.
 155. The compound of claim 146, wherein atleast one modified sugar comprises a 2′-O-methoxyethyl group.
 156. Thecompound of claim 146, wherein the modified oligonucleotide is a gapmer.157. The compound of claim 146, wherein the modified oligonucleotide isa single-stranded modified oligonucleotide.